LIBRARY Michigan State University This is to certify that the dissertation entitled ASPECTS OF RUMINANT TRIGLYCERIDE METABOLISM presented by DAVID LESLIE PULLEN has been accepted towards fulfillment of the requirements for Ph. D. Animal Science degree in Major professor 6 Mom... “2—...- ‘ ‘ '1 m" ' ' ' A 0-127" IVIESI_J RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from 1—;‘—. your record. FINES will be charged if book is returned after the date stamped below. ASPECTS OF RUMINANT TRIGLYCERIDE METABOLISM By David Leslie Pullen A DISSERATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1987 ABSTRACT ASPECTS OF RUMINANT TRIGLYCERIDE METABOLISM By David Leslie Pullen Secretion of triacylglycerol (TG) from ruminant liver slices was low compared to other species. Attempts to stimulate secretion of TG by 1) increasing fatty acid concentration (.ZmM to lmM), 2) comparing saturated to unsaturated long chain fatty acids, 3) adding insulin, estradiol or triiodothyronine, or 4) addition of propionate (lmM) were ineffective. Reducing time from slaughter to incubation (25 min. to 10 min.) did not increase TG secretion rates. Esterification rates were linear with time (up to 8 hours) suggesting tissue slices were viable. Minimal Essential Media (MEM) was a more effective medium than Krebs Ringer Bicarbonate (KRB) at stimulating esterification of fatty acid to T6 in tissue. Hepatic TG secretion from liver slices of cattle, sheep, pigs and a guinea pig (1002 adipose synthesis) was 5 x lower compared to rats and rabbits (50:50; adipose:liver) and 15-20 x lower compared to fish and chickens (1002 liver). The capacity for "de novo" hepatic fatty acid synthesis was highly correlated with the ability to secrete TC in these experimental conditions. David Leslie Pullen Results of a continuous infusion of labelled palmitate in high producing early lactating cows showed that transfer of nonesterified fatty acids (NEFA) into TG increased from 30 to 60 days (7 to 15%, respectively) while oxidation of NEFA to CO2 decreased (from 242 to 15%). Plasma NEFA concentration decreased from 363 to 174 uM and was positively correlated (R=.7) to milk fat production, suggesting direct uptake of NEFA by the mammary gland. Plasma NEFA concentration was positively correlated with NEFA turnover rate (R-.69) Z NEFA converted to 002 (R=.76) and negatively correlated with Z NEFA converted to TG (R=.53). Methionine hydroxy analog (MHA) did not appear to precipitate a lipotrophic response by the liver. A model for hepatic TG production was tested in wethers using a single dose of labelled palmitate. Calculated turnover rates in the microsomal (lipoprotein precursor pool) and slowly turning over "fat droplet" pool were 7.8 mg.h-ikg bwt' and 2.3 mg h-} kg bwt'i, respectively. Turnover rates in the l microsomal pool and fat droplet pool decreased and increased, respectively as Z of TB in liver increased. This is consistent with decreased hepatic TG secretion when NEFA levels are increased such as in early lactation potentiating subsequent development of fatty liver. Half life of endogenous plasma TB averaged about 10 minutes. ACKNOWLEDGEMENTS The author expresses his appreciation and gratitude to his major professor, Dr. Roy Emery, for his patience, direction and guidance throughout this period of study. In addition, the provision of financial support was much appreciated and the study could not have been completed without it. Appreciation is also extended to the members of my committee, Drs. D. R. Romsos, H. Bucholz and W. G. Bergen. Contributions of Drs. J. T. Huber and D. L. Palmquist are also appreciated. Special appreciation is extended to Mr. Jim Liesman for his expertise both in the laboratory and with statistical analysis. I would also like to express thanks to Dr. Kent Ames for his surgical skills and the use of the M.S.U. Veterinary school facilities. The author is grateful to other faculty, staff and especially fellow graduate students in the Animal Science department for their help and advice during this study. Finally, the author wishes to again express his sincere appreciation to his mother for her patient skill in completing the task of typing this manuscript. In addition, the encouragement, support and patience from my wife, Pam, and other members of my family was invaluable. ii DEDICATION The fear of the Lord is the beginning of wisdom: a good understanding have all they that do His commandments: His praise endureth for ever. Psalms 111:10 iii TABLE OF CONTENTS INTRODUCTION . REVIEW OF LITERATURE . Hepatic Fatty Acid Metabolism . . . Fatty Acid Synthesis "de novo" Fatty Acid Oxidation . . Hepatic Lipoprotein Synthesis and Secretion Limiting Factors Hormonal Effects Characteristics of Circulating Lipoproteins Role of Apolipoproteins . . Lipoprotein Catabolism . Contribution to Milk Fat . RUMINANT HEPATIC TRIACYLGLYCEROL SECRETION IN VITRO. Materials and Methods Source of Tissue . Tissue Preparation . . . Triacylglycerol Esterification and Secretion Studies. . . . . Media Preparation Incubation Procedure . . Sample Extraction and Separation . Calculations . . Statistical Analysis Results . Species Comparison . Discussion. . FATTY ACID KINETICS IN LACTATING COWS Materials and Methods . Experimental Procedure . Laboratory Analysis. Calculations . . Statistical Analysis . Results . . . . . Discussion . ENDOGENOUS TURNOVER OF HEPATIC AND PLASMA TRIACYLGLYCEROL IN SHEEP Materials and Methods. iv 1 3 A ’) 5 15 16 18 25 31 36 38 42 44 44 44 47 47 48 49 49 51 51 52 67 78 93 94 96 97 99 100 101 117 126 127 Ewe Study . Wether Study . . . . . . Radiochemicals/Chemicals Laboratory Analysis . Liver Fractionation Calculations Results . . . . . . . . . . . . . . . . . . . Endogenous Intestinal Lymph Production Endogenous TG Turnover Study Discussion Endogenous TG Turnover Study SUMMARY AND CONCLUSIONS APPENDICES BIBLIOGRAPHY Endogenous Intestinal Lymph Production. 127 128 129 130 130 132 133 133 133 145 145 146 153 157 161 10. 11. 12. LIST OF TABLES Composition of the Major Bovine Lipoprotein Classes Esterification and Secretion of Fatty Acid as TC in Liver Slices from a Female Nonpregnant Rat . Esterification and Secretion of Fatty Acid as TC in Ovine Liver Slices (Ewes I and II).. Effect of Added Propionate (1.0 mM) on Esterification and Secretion of Fatty Acid as TC in Steer Liver Slices (Steers I and II). . The Effects of Krebs-Ringer (KRB) vs Minimal Essential Media (MEM) with Serum or Bovine Serum Albumin (BSA) on Esterification and Secretion of Fatty Acid as TG in Liver Slices of a Steer (Steer IV). Effect of Combination of Fatty Acids vs Palmitate Alone as Substrate for TG Esterification and Secretion in Steer Liver Slices . Effect of Fatty Acid Concentration .2mM vs lmM on Esterification and Secretion of Fatty Acid as TC in Lamb Liver Slices. Effect of Time from Slaughter to First Flask Incubation on Esterification and Secretion of Fatty Acid as TC in Rat Liver Slices. . . . . . Effect of Decreased Time from Slaughter to First Flask Incubation on Esterification and Secretion of Fatty Acid as TC in Steer Liver Slices. Effect of Preincubation Holding Temperature (2°C vs 25°C) in Esterification and Secretion of Fatty Acid as TC in Steer Liver Slices . . . . . . . . . . Effect of Insulin (I) and Puromycin (P) on Esterification and Secretion of Fatty Acid as TG from Liver Slices of a Rat . . Effect of Insulin (I), Estradiol (E) and Oleic (0) Acid on Esterification and Secretion of Fatty Acid as TG from Heifer Liver Slices over Six Hours of Incubation . vi 33 52 54 . 54 - 55 - 56 - 57 - 59 . 59 .60 ‘63 ~65 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Effect of Insulin (I), Estrogen (E) and Triiodothyronine (T ) on Esterification and Secretion of Fatty Acid as sG from Lamb Liver Slices over Six Hours of Incubation . . . . . A Comparison of Fatty Acid Esterification and Secretion as TC in Esterification and in Liver Slices of Esterification and in Liver Slices of Esterification and in Liver Slices of Esterification and in Liver Slices of Laying Hens Esterification and in Liver Slices of Esterification and in Liver Slices of Esterification and in Liver Slices of Rat vs Pig Secretion of Fatty Acid as a Pig . . . . . Secretion of Fatty Acid as a Guinea Pig . . Secretion of Fatty Acid as Rabbits . . . . . . Secretion of Fatty Acid as Single Comb White Leghorn Secretion of Fatty Acid as Trout . . . . . Secretion of Fatty Acid as a Functional Nonruminant Secretion of Fatty Acid as a Neonatal Lamb . . . Composition of Concentrate Fed to Cows Individual Observations on Lactating Cows Effect of MHA on Production of Milk and Milk Fat . Effect of MHA on Plasma NEFA and TG Measurements . Effect of Stage of Lactation on Plasma NEFA and TG Measurements Endogenous Triglyceride (TG) Production by the Intestine Relative Liver Measurements from Biopsied Wethers Plasma TG Kinetics Concentration of Plasma Nonesterified Fatty Acids in Wethers (NEFA) throughout the Experiment Liver TG Kinetics in Wethers . vii TC TC TC TC TC TC TC 66 69 70 70 72 72 73 75 75 95 102 104 104 107 134 139 140 140 144 Appendix Tables 1. Composition of Minimal Essential Media (MEM) with Earle' s Salts and 25 nM Hepes Buffer and without L- -glutamine . . . . . . Media Extraction Procedure In_Vitro Assay . Summary of HPLC Procedure . Composition of Krebs- -Ringer Buffer (KRB) and Minimal Essential Media (MEM) viii 158 159 160 161 10. 11. 12. LIST OF FIGURES Effect of insulin and puromycin on hepatic secretion of fatty acid as TG from rat liver slices . . . Relative capacity for hepatic lipogenesis versus hepatic secretion of fatty acid as TG . Milk fat production versus plasma NEFA concentration . . Effect of stage of lactation (30, 60 and 90 D) on plasma NEFA concentration . . . . Entry rate of palmitate in fed lactating dairy cows . . . . . . . . . . Percent of NEFA turnover incorporated into TG at various plasma NEFA concentrations . Percent of NEFA turnover oxidized to C at various plasma NEFA concentrations Plasma specific activity of NEFA (dpm/umole) and TG (dpm/umole TGFA) in MHA XI . . Specific Activity (S.A.) of TC in ewe IV . Representative figure of hepatic and plasma TG kinetics in a wether (#185) Relationship between percent TC in the liver and turnover rate (tr) . . . . . . . A tentative model for endogenous TG turnover in the liver and plasma of the wether ix 62 76 106 108 111 113 115 123 135 136 114 151 INTRODUCTION The liver plays a critical role in the regulation of lipid metabolism in all species. Most of the current knowledge of hepatic lipid metabolism has been derived from studies with laboratory rats. Results obtained with laboratory rats are often extrapolated to other species and assumed to Operate in like manner. Unfortunately, invalid interpretations may result~ from such application to ruminant species. This should not be altogether unexpected considering the differences in the anatomy of the gastro- intestinal tract and consequent ruminant mode of ingestion and digestion. As a consequence of these dietary differences, intermediate metabolic events are also affected. For example, the major site of "de novo" fatty acid synthesis in the ruminant is adipose, whereas in the rat, it is split between liver and adipose. Also in the ruminant, gluconeogenesis occurs continuously both as a result of low dietary glucose and its high demand for lactation. In the rat, however, dietary glucose is sufficient to limit endogenous gluconeo- genesis except during fasting and post-absorptive states. Gluconeogenesis and lipogenesis are processes which compete for ATP and carbon skeletons and thus cannot occur simultaneously at maximal rates in the same organ. Limited hepatic lipid synthesis also ensures that acetate, a major by product of rumen fermentation, will pass from 2 the portal blood to the peripheral blood with little intermediary metabolism by the liver. This enables acetate, an important energy substrate, to be available for metabolism by extrahepatic tissues and glucose can be spared for synthesis of lactose. In both species, the liver is a major site of fatty acid esterification. Factors controlling the subsequent fate of fatty acid esterified to triacylglycerol have not been clearly elucidated. The extent to which hepatic triacylglycerol is incorporated into lipoprotein and secreted as very low density lipoprotein (VLDL) has been widely studied in rats and chickens, but no information is available in regards to the importance of this pathway in ruminants. Hepatic VLDL secretion is believed to be a major contributor to milk fat production in early lactation in the lactating cow. This study attempts to define qualitatively the role of the ruminant liver in secretion of fatty acid as VLDL. This was accomplished by designing an in zitgg system for measuring secretion of TG from.liver slices of ruminants and for purposes of comparison, a number of other species. In addition, the contribution of fatty acid to hepatic VLDL was also measured in lactating dairy cows during the first two months of lactation. Consideration is also given to the potential contribution of hepatic VLDL to production of milk fat. Finally, attempts to measure TG turnover in the liver and plasma compartments are also described in a study with mature nonpregnant ewes. REVIEW OF LITERATURE Upon initiation of lactation, the high producing dairy cow partitions much of her energy flow to the mammary gland to meet the needs of the rapid onset of milk production. Approximately one-half of the fatty acids secreted in milk fat are synthesized "de novo" in the mammary gland. The majority of this half arises from acetate with one-sixth coming from B-hydroxybutyrate (Palmquist and Mattos, 1978). The long chain fatty acids (16 and 18 carbons) in blood contribute the other half of the milk fat to the mammary gland and are obtained by the action of lipoprotein lipase (LPL) on blood triacylglycerol (TG) at the mammary gland (Scow et al., 1973; Barry et al., 1963; Lascelles et al., 1964). Intestinal chylomicron and very low density lipoprotein (VLDL) synthesis (exogenous) or hepatic synthesis of VLDL from nonesterified fatty acid (NEFA) (endogenous) are the presumed sources of NEFA substrate for the mammary gland (Palmquist and Mattos, 1978). Adipose tissue lipolysis or action of LPL on lipoproteins in early lactation, provide most of the NEFA for hepatic VLDL production which is assumed to be an active mechanism.in the cow during the first three to four weeks of lactation (Puppione et al., 1973). However, no studies have addressed specifically hepatic VLDL contribution to the mammary gland in the bovine. Hepatic Fatty Acid Metabolism The hydrophobic nature of long chain NEFA requires that they be transported in the blood complexed to protein. Albumin has been shown to be the major vehicle for NEFA transport through the blood (Dole, 1956; Gordon and Cherkes, 1956). In fact, when albumin is added, the concentration of NEFA.may be increased up to 500 times more than the maximum solubility in a similar solution of salt formed to mimic plasma. Results of Spector et al., (1969) suggest that bovine serum albumin (BSA) contains six high energy binding sites for NEFA, including the primary and secondary binding sites, and an additional 63 possible tertiary binding sites. In general, the 016:0 NEFA are more tightly bound than 018:0, 18:1, 18:2, 14:0 or C12:0. Association of NEFA with albumin appears to involve an electrostatic attraction of the carboxyl group on the NEFA to cationic sites on the protein, together with hydrophobic interactions between the hydrocarbon tail of the NEFA and non-polar side chains of the protein (Spector et al., 1969). As adipose TG hydrolysis occurs, NEFA are released and bind to bovine serum.a1bumin (BSA) for transport in the blood. There is considerable evidence that suggests uptake of NEFA by the liver is concentration dependent. This has been observed in rats (Heimberg et al., 1974) and in ruminants (Katz and Bergman, 1969b) up to concentrations of 2-3mM. Weisiger et al., (1981) demonstrated the existence of a putative receptor for hepatic uptake of albumin bound fatty acids by the liver. In addition, the interaction of 5 albumin with the putative albumin receptor was not influenced by the number of fatty acid molecules bound to the individual albumin molecules. Thus, as the number of fatty acids bound to albumin increased, there was a proportional increase in fatty acid available to be taken up by the hepatic albumin receptor. Saturation occurred only when the availability of receptors became limiting. They further concluded that the data excludes the possibility of unbound and total concentrations of fatty acids as principle determinants of uptake. When oleate was increased with BSA constant, uptake and concentration of unbound oleate were not correlated. In addition, oleate uptake was diminished when albumin was added. In contrast, Abumrad et al., (1981 and 1984) found that the kinetic constants for permeation in adipocytes were the same whether the concentration of unbound fatty acid was varied by changing total fatty acid at a constant BSA level or by varying BSA while keeping total fatty acid constant. This suggests there may be differences between cell types. Bergman et al., (1971) determined that the liver of conscious fed sheep extracts about 252 of all NEFA entering the blood stream. Others have shown that the fractional clearance rate of NEFA.in sheep remains constant at about 102 over a range of physiological states and arterial NEFA concentrations (Thompson and Darling, 1975 and Thompson et al., 1978). Several reports in the lactating dairy cow suggest fractional clearance rate to be around 162 increasing to about 202 after fasting up to six days (Lomax and Baird, 1983; Reid et al., 6 1979). Fractional clearance rates for NEFA in livers of nonruminants appear to be somewhat higher, 402 to 502 in isolated perfused rat livers (Mayes, 1970; Soler-Argilaga et al., 1971). Increasing NEFA perfusate concentrations from .36 qu/ml to 3.97 qu/ml resulted in increased uptake of NEFA from 402 to 802 (Nestel and Steinberg, 1963). Once the NEFA enter the cytosol, they are bound by a 12,000 dalton fatty acid binding protein (FABP). This has been demonstrated in a number of species (including rat, human, avian and bovine) and tissues (liver, adipose, muscle, and intestine) (Ockner and Manning, 1974; Wu-Rideout et al., 1976; Haq et al., 1982; Ockner, 1972; Katongle and March, 1979; and Smith et al., 1985). The role of FABP is unclear. It may in part function to transfer the hydrophobic NEFA to the site of acylation between microsomes and the outer mitochondrial membranes (wu-Rideout et al., 1976). Ockner and Manning,(1974) observed an increase in the concentration of intestinal FABP when a high fat diet was fed. This may suggest a partition of cellular NEFA toward esterification, preventing accumulation within the cell. Wu-Rideout et al., (1976) showed that fatty acid activation was stimulated by addition of FABP to hepatic microsomes in fed rats. In contrast, FABP was shown to inhibit mitochondrial fatty acid activation. Whereas addition of flavaspidic acid, a compound which competes with NEFA for binding sites on FABP, reversed the effects on mitochondrial fatty acid activation in the presence of FABP. They interpreted this to mean that FABP specifically enhances the flow of NEFA toward microsomal 7 esterification and subsequent TG formation. When FABP binding sites were filled, they were directed toward oxidation. In addition, FABP was elevated in rats fed cholestyramine, a compound which increases hepatic TG synthesis, (Kempen et al., 1983) and decreased in rats fed diets high in linoleic acid (Herzberg and Rogerson, 1981). The activity of FABP in bovine liver was shown to be about 102 of that in rat liver (Smith et al., 1985). It is difficult to discern whether this represents a species difference or variation in assay or diet. Indeed, it may reflect the low level of fatty acid synthesis "de novo" in ruminant livers. In liver, as in all mammalian tissues, fatty acids must be activated by conversion to fatty acyl CoA before further metabolism can occur. This activation is catalyzed by different categories of acyl CoA synthetases broadly classified for short, medium and long chain fatty acids (Bell, 1979). In ruminants, activity of the short and medium chain fatty acyl synthetases has received much more attention than the long chain acyl CoA synthetase. In nonruminant liver, long chain acyl CoA appears to be localized in the microsomes and on the outer mitochondrial membrane (Groot et al., 1976). Whether or not the specific location of these synthetases commits the fatty acid to a particular pathway i.e. outer mitochondrial membrane to oxidation and microsomes to esterification, is not known. Several enzymes involved in glycerolipid synthesis have a similar distribution and some (eg. phosphatidate phosphohydrolase) are found in the soluble 8 fraction of the cell (Zammit, 1984). The hepatic activity of the synthetase has been shown to be much greater than the rates of B-oxidation in the mitochondria (Pande, 1971) and esterification with alpha-glycerolphosphate in microsomes (Lloyd-Davies and Brindley, 1973) suggesting that this enzyme is not rate limiting for subsequent metabolism of the fatty acid. At this point, the fatty acyl CoA may be diverted to oxidation and possibly ketogenesis in the mitochondria or alternatively to esterification to T6, phospholipid (PL) or cholesterol ester (CE) within the microsomes. In ruminants, however, the hepatic formation of CE is limited (Payne and Masters, 1971) and thought to occur primarily in the plasma lecithin-cholesterol acyltransferase (LCAT) system (Noble et al., 1975). The eventual pathway to which the fatty acyl CoA is diverted depends on physiological state. Factors regulating this diversion will not be considered at great length in this review. Recent comprehensive reviews on this process are available (Zammit, 1984; Haagsman and VanGolde, 1984). i The process of esterification appears to operate much the same in ruminants as it does in nonruminants. Fatty acids are esterified by the glycerol phosphate pathway as well as by the glyceraldehyde 3-phosphate and dihydroxyacetone phosphate pathway in the rat (Rao, et al., 1968). Benson and Emery, (1971) suggested the existence of an alternative pathway for hepatic palmitate esterification when no glycerol phosphate was added to the incubation medium of bovine liver homogenates. The general pathway for esterification 9 begins with glycerol phosphate acyltransferase (GPAT) or the enzyme dihydroxyacetone phosphate acyltransferase (DHAPAT). It has been suggested that these microsomal enzyme activities might reside in the same enzyme protein (Schlossman and Bell, 1977). Dihydroxyacetone phosphate acyltransferase has also been found in rat liver peroxisomes (Hajra et al., 1979). The resulting products of the first acylation, 1-acyl glycerol 3-phosphate and l-acyl hydroxyacetone phosphate are interconvertible via an NADP- linked reductase and the eventual product, i.e. phosphatidic acid (PA) is formed exclusively through the action of lysophosphatidate acyltransferase on 1-acylg1ycerol 3- phosphate (Zammit, 1984). Activity of GPAT and DHAPAT has also been reported in mitochondria at low levels, the major product at this site being lysophosphatidate. These mitochondrial enzymes are located in the outer membrane and the active site faces the intermembrane space (Nimmo, 1979). The physiological implications for this location may be related to removal of long chain acyl CoA from the intermembrane space, particularly under conditions in which acyl CoA utilization for acylcarnitine synthesis is required to be maintained at a minimum (Zammit, 1984). The next step in the pathway involves the conversion of PA to diacylglycerol by the enzyme phosphatidate phosphohydrolase (PAP) or alternatively by CTP phosphatidate cytidylyl-transferase to CDPzdiacylglycerol (Haagsman and VanGolde, 1984). The activity of PAP is found in the soluble and membrane fractions of liver homogenates (Jelsema and 10 Morre, 1978), and is considered to catalyze the rate limiting step in TG synthesis because this enzyme shows the lowest activity of the enzymes that convert g1ycerol-3-phosphate into TG (Fallon et al., 1977; Brindley, 1978). In addition, under varying conditions the activity of PAP appears to change several fold in the same direction as changes in acylglyceride synthesis, particularly TG synthesis (Lamb and McCue, 1983). However, this has not been a consistent observation (Pikkukangas et a1, 1982). In diabetes and metabolic stress, the observed increase in hepatic TG esterification and secretion appears to be related to the high activities of PAP (Brindley and Sturton, 1982; Brindley and Lawson, 1983). This occurs because of the glucocorticoid stimulated synthesis of PAP which increases the potential of the liver to synthesize TG. This potential is expressed when fatty acids mobilized frmm adipose are made available as a result of the high concentrations of glucagon, adrenaline and corticotropin relative to insulin (Pittner et al., 1985). Elevated concentrations of fatty acids stimulate PAP activity in liver (Lamb and McCue, 1983; Cascales et al., 1984) and also cause the functional activation of PAP by promoting its translocation from the cytosol of the hepatocytes to the membranes on which PA is being synthesized (Cascales- et al., 1984; Butterwith et al., 1984). Pittner et al., (1985) investigating a coordinated control of TG synthesis and gluconeogenesis (GNG) in stress conditions demonstrated that the high concentrations of cAMP that occur in the liver 11 in stress and diabetes, could augment the glucocorticoid effect in increasing the total PAP activity and the potential for TG synthesis. Cyclic AMP normally translocates PAP from the membrane to the cytosol (Butterwith et al., 1984) and in fact inhibits TG synthesis (Pelech et al., 1983). However, at high fatty acid concentrations which counteract the CAMP displacing effect on PAP, this cytosolic resevoir of potential PAP activity can be expressed (Butterwith et al., 1984). Thus, the increased PAP activity can be realized by translocation to the membrane compartment to match the increased supply of fatty acids that is mobilized from adipose during stress or diabetes with resultant increases in TO synthesis (Pittner et al., 1985). The last step in esterification occurs as diacylglycerol (DC) is acylated by diacylglycerol acyltransferase to TG. Alternatively, the DG may be acylated by a choline or ethanolamine phosphotransferase to form phospholipids (PL) (Haagsman and VanGolde, 1984). The DC acyltransferase is located solely in the microsomal fraction (Wilgram and Kennedy, 1963). Differential incorporation of fatty acids into TG and PL suggest some form of regulation at the DG branchpoint. Stearic and linoleic acids were preferentially esterified into PL and palmitic into TC in bovine liver homogenates (Benson and Emery, 1971). These studies suggested that chain length and degree of unsaturation may influence incorporation into acylglycerides. Similar results were observed in sheep with linoleic being incorporated into PL predominantly (Payne and Masters, 1971). Physiological state 12 will also influence incorporation. Fatty acids stimulate TC synthesis whereas PL synthesis is hardly affected. Hepatic TG synthesis was greatly increased over PL synthesis (content and composition unchanged) in cows with hepatic lipidosis induced by fasting or displaced abomasum (Brumby et al., 1975; Herdt et al., 1983). This would suggest a hypertrophy of lipid droplets within the cell which would not require equivalent increases in PL to surround the lipid core. Fatty Acid Synthesis Another potential source of fatty acid substrate for T6 esterification is hepatic fatty acid synthesis. This pathway is of little consequence in the ruminant liver, however, and therefore not likely to contribute much to lipogenesis in the lactating cow (Ballard et al., 1969). Deficiency of fatty acid synthesis in bovine liver was first established by Hanson and Ballard,(l967) who attributed it to low levels of ATP citrate lyase and NADP malate dehydro- genase in sheep and cows. (Others have shown as well, that glucose carbon is not lipogenic in the perfused mammary gland (Hardwick, 1966) or in adipose tissue (Hanson and Ballard, 1967). The limited role of glucose in fatty acid synthesis is a reflection of the low level of dietary carbohydrate that appears as glucose in the circulation and the cows attempt to conserve glucose for production of lactose during lactation. The fetal ruminant, on the other hand, has a maternal supply of glucose and resembles the nonruminant in that it does have limited ability to produce 13 fatty acids from glucose in liver (Hanson and Ballard, 1968). The activity of ATP-citrate lyase and NADP-malate dehydrogenase is increased substantially in the fetal liver compared to the adult ruminant, although levels are still much lower than in the fetal or adult rat liver. The possibility that glucose supply was limiting activity of the citrate cleavage pathway has also been tested. Ballard et al., (1972) fed a high carbohydrate diet, or infused abomasally or intravenously, glucose in young sheep for several weeks. Activities of ATP citrate lyase and NADP malate dehydrogenase were increased from low values but they were variable and even the highest values were much lower than in the rat. The synthesis of fatty acids in the liver requires an extramitochondrial source of acetyl CoA. In the ruminant, acetate is a major product of the carbohydrate fermentation in the rumen thus providing a potential source of carbon for "de novo" fatty acid synthesis. However, many studies have shown that very little acetate is actually taken up by liver 13 33533 (Leng and Annison, 1963; McCarthy et al., 1958) or in gizg (Baird et al., 1975; Bergman and Wolff, 1971; Cook and Miller, 1965). In addition, low activities of acetyl CoA synthetase have been found in livers of sheep and cattle (Cook et al., 1969; Hanson and Ballard, 1967; Knowles et al., 1974), and in the presence-of propionate or butyrate what little activity is present is greatly diminished (Ash and Baird, 1973). In cattle, acetyl CoA synthetase was found predominantly in the mitochondria (Quraishi and Cook, 1972) 14 with low cytosolic activity (Ricks and Cook, 1981). Considering the lack of citrate cleavage enzyme, this would very likely not provide a source of extramitochondrial acetyl CoA. Fatty acid synthesis also requires a source of reducing equivalents. In nonruminants, this is met by NADPH from the pentose phosphate pathway or from the reductive decarboxylation of malate via the citrate cleavage pathway (Bell, 1979). As mentioned previously, the activity of NADP malate dehydrogenase is low in ruminants. It has been suggested that the little fatty acid synthesis that does occur utilizes NADPH from the pentose phosphate pathway (Ballard et al., 1969) as significant hepatic activity of these enzymes have been detected in sheep and cattle (Hanson and Ballard, 1967; Filsell et al., 1963; Raggi et al., 1961). In summary, it appears that virtually all of the ruminally derived acetate passes through the liver without being metabolised. The physiological basis for this could be: 1) to ensure that acetate, an important extrahepatic energy substrate, is made available to peripheral tissues (e.g. mammary gland for milk fat synthesis) which is especially important when glucose demand is high, or alternatively, 2) it ensures that very little fatty acid synthesis will occur in the cytosolic fraction of ruminant liver from acetate which in turn will limit the competition that would result between lipogenesis and gluconeogenesis for ATP and carbon skeletons at the same time in the liver (Ricks and Cook, 1981).. 15 Fatty Acid Oxidation The other major route that fatty acyl CoA may follow is oxidation. Very few papers have dealt specifically with hepatic long chain fatty acid oxidation in ruminant livers. It is generally accepted that beta-oxidation of long chain fatty acids in tissues is controlled by their availability. As plasma concentration is increased, as in times of metabolic stress, fatty acid oxidation is increased. Pethick et al., (1983) demonstrated a linear relationship between plasma NEFA concentration and the rates of entry and oxidation. The primary site of beta-oxidation and ketogenesis is within the mitochondria. The peroxisomes also contribute small amounts estimated to be a minimum of 62 to 72 of total oxidation in bovine liver (Jesse, 1984). In order for beta-oxidation to occur, fatty acyl CoA must be translocated from the cytosol to the mitochondria because the inner mitochondrial membrane is impermeable to long chain acyl CoA derivatives. Fatty acyl CoA is converted to fatty acyl carnitine by carnitine acyl transferase I (CATI) and back to fatty acyl CoA by carnitine acyltransferase II (CATII). These two enzymes are located on the inner and outer surface of the inner mitochondrial membrane. Within the mitochondria, the fatty acyl CoA may then undergo beta- oxidation. The capacity of the ruminant liver to oxidize long chain fatty acids appears to be relatively low (Bell, 1979) and the mechanisms controlling oxidation have not been clearly elucidated. Several recent reviews of the subject 16 are available both for nonruminants (Zammit, 1984) and ruminants (Jesse, 1984). Hepatic Lipoprotein Synthesis and Secretion Very little is known about the mechanisms governing the control of lipoprotein synthesis and secretion from the liver. Understanding what controls the association of the lipid and protein components, their subsequent transport through the cell and final secretion into the blood is one of the greatest challenges in this multidisciplinary area of cell biology, molecular biology and biochemistry. The lipoprotein particle is composed of a neutral lipid center, containing TG and cholesterol esters (CE) in association with phospholipid (PL), cholesterol and protein which surround the central hydrophobic core. The particle is designed to transport lipid materials through the blood for storage and utilization by extrahepatic tissues. Hepatic TG fatty acids are the immediate precursors of the TG found in very low density lipoprotein (VLDL) (Havel et al., 1962). There appears to be two precursor pools of TG fatty acid substrate within the liver: 1) a large TG pool identified as "floating fat" and 2) a smaller pool identified as the "microsomal" pool (Mayes, 1976). When labelled free fatty acids are taken up by the liver, the small microsomal pool has a much higher specific activity than the larger pool of floating fat (Stein and Shapiro, 1959; Havel et al., 1962; Baker and Schotz, 1967). Topping and Mayes,(1982) demonstrated, using perfused rat livers, that the specific activity of the VLDL TG fatty acid was 702 of the specific 17 activity of NEFA while that of the hepatic TG fatty acids (mainly floating fat) was 102. It has also been demonstrated that lipolysis of floating fat results in re-esterification in the microsomal fraction (Bar-On et al., 1971). The existence of two pools of TG fatty acid and the relationship between them in terms of VLDL secretion are consistent with the secretion model described by Zech et al., (1979). This model suggests the existence of a rapidly turning over pool of TG and a much more slowly turning over pool, (this will be discussed in more detail later), both of which contribute to plasma VLDL. Electron microscoPic studies in perfused livers of rats have revealed micrographs of particle movement through the hepatocyte with eventual secretion of a lipoprotein particle (Jones et al., 1967). More recent studies involving cytoimmunochemical techniques (Alexander et al., 1976) and kinetic approaches (Janero and Lane, 1983) have been utilized in an attempt to define the sequence of events that leads to release of mature VLDL particles. A combination of the above approaches has resulted in the following pr0posed 'scheme for VLDL formation. Initiation of VLDL apolipoprotein translation occurs at the rough endoplasmic reticulum (RER) followed by ejection into the RER lumen near the junction with the smooth endoplasmic reticulum (SER). During this time period (about ten minutes) the apolipoproteins are associated with some of the phosphoglycerides (especially phosphatidylcholine) and core glycosylation occurs (Janero 18 and Lane, 1983). The apolipoproteins are then assembled with TG some ten minutes later to form a lipoprotein particle. The lag period between formation of the apolipoprotein particle and subsequent association with TG may reflect the transit time required for movement of the apolipoprotein from the RER to the SER where TG, CE and cholesterol are incorporated into the lipoprotein particle. The core VLDL particle is then budded off from the SER and is transported to the Golgi where they reside in secretory vesicles (Marsh, 1984). Additional PL is added at the Golgi (possibly additional CE and cholesterol are also added) and the secretory vesicles containing the nascent lipoprotein particles travel to the plasma membrane via the microtubular/microfilament system (Drevon et al., 1984; Vance and Vance, 1985). The VLDL particles fuse with the plasma membrane and are released into the extracellular space (i.e. Space of Disse) and finally enter the circulation. The whole process takes about twenty to thirty minutes as ascertained in zitgg (Janero and Lane, 1983) and in 3132 (Baker and Schotz, 1964; Havel and Goldfien, 1961). Limiting Factors Due to the complicated nature of this pathway, it is obvious that there are many factors involved in coordinating the eventual formation and secretion of the mature hepatic VLDL. Among these factors are diet (as it affects substrate availability, ie. PL, NEFA, CE, cholesterol and protein), and hormonal regulation, related to physiological state and sex. Regarding substrate availability, PL, more specifically 19 phosphatidylcholine (PC), has been shown to be required for lipoprotein secretion. A significant reduction in hepatic PL content observed in cows with severe hepatic lipidosis has been implicated in reduced hepatic TG secretion (Herdt et al., 1983). Studies by Best and Huntsman,(l932) revealed that fat deposition in rat liver could be prevented by addition of lecithin to the diet and further that choline could be substituted for lecithin. Other researchers have shown that a choline deficiency results in decreased plasma lipoproteins and accumulation of hepatic TG (Kuksis and Mookerjea, 1978; Margolis and Capuzzi, 1972). In choline deficient rats, a decrease in hepatic PC (from 63 to 48 umol/lOOg body weight) was accompanied by an increase in phosphatidyl ethanolamine (PE) (from 18 to 40 umol/lOOg body weight) (Tokamkjian and Haines, 1979). The observed increase in PE, however, does not prevent the reduced lipoprotein output and increased accumulation of hepatic TG (Tokamkjian and Haines, 1979). An alternative pathway for PC synthesis is via methylation of PE (amounting to 202 to 402 of total PC synthesis in normal rat liver) (Sundler and Akesson, 1975). In fact, the activity of this pathway is almost doubled in a choline deficiency (Schneider and Vance, 1978). Whether or not this occurs ip’yiyg has not been demonstrated. In any case, this pathway does not appear to override the effects of a choline deficiency nor it is necessary for lipoprotein secretion (Vance and Vance, 1985). A deficiency in inositol, a precursor for phosphatidyl inositol (PI) has also been associated with fatty liver 20 (Reed et al., 1968). Additionally liver inositol and liver fat were reported to be inversely related in the bovine (Gerloff et al., 1981). However, supplemental inositol given as myo-inositol to dairy cows in early lactation did not appear to have any effect on liver TG or total liver inositol (Gerloff et al., 1984). Myo-inositol has been shown to be an effective lipotrophic agent in several other species (Holub, 1982). Availability of NEFA is obviously very important to VLDL-TG secretion as NEFA are the precursors for hepatic VLDL- TG. Triglyceride output by perfused livers from normal fed rats was shown to be proportional to both concentration of NEFA in the medium and to total uptake of NEFA by the liver (Heimberg et al., 1974). These authors also showed that TG secretion was a saturable process. Uptake and metabolism.of NEFA by the perfused liver was increased in proportion to NEFA concentration, however, the capacity of the liver to secrete TG was comparatively less. Triglyceride secretion by normal fed male rats in 31532 had an apparent Vmax of 50-60 umoles/g liver/4 hours (equivalent to 400-500 umoles/liver during the 4 hour period). This corresponded to a steady state concentration of .70-.75mM NEFA (oleate) in the perfusate medium. The apparent Ks (NEFA concentration at half maximal velocity) corresponded to a concentration of .25-.30mM oleate in the perfusate. This concentration is consistent with that observed in man and other animals in a normal fed state. Mayes and Felts,(1967) showed that the percent of NEFA secreted as VLDL from perfused livers of fed 21 rats, was decreased as perfusate concentration of NEFA was increased from .3mM to .9mM and finally to 1.9mM. The excess NEFA was diverted to storage and oxidative pathways. The relationship between plasma NEFA concentration and hepatic TG secretion has important implications in bovine metabolism in early lactation. The rapid mobilization of NEFA from adipose stores often results in development of varying degrees of hepatic lipidosis (HL). For a detailed description of the etiology of HL in bovine, man and other domestic species, see review of Gerloff,(1985). Studies using fasted cows, which develop HL, have shown a reduced concentration of TG rich lipoproteins (Brumby et al., 1975) and a direct reduction in hepatic TG secretion (Reid et al., 1979). In cows with HL due to a displaced abomasum, there was a reduction in the serum dextran sulphate precipitable lipoprotein fraction (DSP) (TG rich fraction) (Herdt, et al., 1983). In contrast to results of Brumby et al., (1975) and Reid et al., (1979), this study observed an increase in serum.TG in fasted cows vs control, suggesting fasted cows may not always be suitable models for HL. Results indicate that bovine postpartum HL is accompanied by a decrease in TG secretion (Herdt et al., 1983). The chemical form of the fatty acid will also affect secretion of TG in rats. Short chain fatty acids (eg. caproic-6C; caprylic-8C) did not stimulate TG secretion, whereas increasing chain length of saturated fatty acids (Lauric 12C, palmitic 16C, stearic 18C) increased TG secretion proportionately. However, TG secretion was inversely related 22 to the number of double bounds in 18 carbon fatty acids (Kohout et al., 1971). More recently, however, others have found that addition of oleic or linoleic acid to perfusate medium increased TG secretion over that of palmitate (Goh and Heimberg, 1973; Goh and Heimberg, 1977; Forte, 1984). Additionally, VLDL formed from oleate were larger and contained less PL and cholesterol per molecule of TG than VLDL from palmitate (Heimberg and Wilcox, 1972). Clearly, hepatic fatty acid synthesis also will contribute to TG secretion although it is not obligatory in rats (Heimberg et al., 1974). Windmueller and Spaeth,(l967) showed that TG secretion was highly correlated with rates of hepatic lipogenesis in perfused livers of fed rats. However, lipogenesis is inhibited as plasma NEFA concentration increases thus contribution of "de novo" fatty acids would decrease (Mayes and Topping, 1974). Fructose (compared to glucose) has also been shown to stimulate TG secretion presumably by increasing hepatic fatty acid synthesis (Topping and Mayes, 1972). Factors affecting the intracellular redox state may also affect VLDL secretion (Mayes, 1976). A more reduced redox state may reduce pyruvate dehydrogenase activity facilitating the switch from carbohydrate to fatty acid oxidation. This would result in a decrease in esterification and in turn TG secretion. This may be why livers from fed rats esterified and secreted more TG than livers from fasted rats which favored oxidation (Mayes, 1976). 23 A few studies have been conducted on the effect of cholesterol feeding on secretion of VLDL from hepatocytes (Dolphin, 1981; Davis et al., 1982). These studies showed a marked increase in CE content of VLDL in cholesterol fed rats (from .03 in the control to .13 ug/mg cell protein in cholesterol fed rats). Addition of oleate to perfusion medium stimulated activity of hepatic microsomal 3-hydroxy 3-methyl glutaryl CoA compared to palmitate (Goh and Heimberg, 1977). In addition, the ratio of cholesterol and PL to TG was decreased with unsaturated fatty acids compared to saturated. Thus, it would appear that the supply of cholesterol and CE alters composition of the VLDL core (Davis et al., 1982). The role of the apoproteins has become a very important subject of study in recent years. In fact, they are obligatory to lipoprotein metabolism in general as they seem to be the signals for specific responses of most, if not all, tissues involved in lipoprotein metabolism. Studies conducted on human patients with abetalipoproteinemia, an inherited disease, have implicated both molecular weight forms of apo-B as a requirement for the secretion of VLDL and chylomicron TG (Herbert et al., 1983). Patients with normotriglyceridemic abetalipoproteinemia (specific deletion of apo B-100) are still capable of assembling and secreting TG rich lipoproteins (Malloy et al., 1981). Secretion in this case presumably arises from the intestines as the low molecular weight form (apo B-48) is of intestinal origin, whereas the high molecular weight form (apo B-100) is of hepatic origin in humans (Edge et al., 1983). 24 Rat liver, however, secretes both forms of apo B although they appear to be independently regulated. Fasting resulted in a selective decrease in apo B-48 (Davis et al., 1985; Marsh and Sparks, 1982). The results of Davis et al., (1985) showed a 502 decrease in secretion of apo B-48 in hepatocytes from (3d) fasted rats. Except for the last time period (8 hours) there was no significant difference in the cellular content of either apo B-100 or apo B-48. Apo E secretion, on the other hand, was stimulated 2-4 fold. Most of this was not associated with the TG secretion. The increase in apo E secretion was consistent with the increased concentration of high density lip0protein E (+622) found in the serum of fasted rats (Davis et al., 1985). In cows, following a starvation induced ketosis, the volume of the rough endoplasmic reticulum was decreased markedly (Brumby et al., 1975). The authors proposed that this may indicate a decrease in apoprotein synthesis which would decrease VLDL output and in turn contribute to the fatty liver observed in bovine liver. In addition, the apo B-48 appears to be secreted in a fonm which contains phosphorylated serine residues, whereas this is absent in the apo B—100 (Davis et al., 1984). These authors suggested that the phosphorylation mechanism.may serve to shuttle VLDL through the cell during its assembly and secretion. In chloroplasts protein phosphorylation is believed necessary for lateral movement in membranes (Staehelin and Arntzen, 1983). Phosophorylation appears to 25 be a general characteristic of a specific class of amphipathic proteins including casein, vitellogenin and myelin basic protein. Clearly, the role of apoproteins in hepatic lipoprotein secretion and eventual distribution is important and a more detailed discussion of the specific effects of the individual apoproteins will follow in a later section. Hggmonal Effects A Another important aspect of hepatic VLDL formation and secretion pertains to the regulatory role of hormones. Obviously, the hormonal effects do not operate in an isolated manner, but are superimposed on fairly unique metabolic conditions such as diet, physiological state, assay conditions, hormone interactions, etc. Therefore, care is required in defining the systems to evaluate hormonal effects in 31532 and i§_yizg and in making general conclusions about specific hormones. This can be seen in the case of insulin where several investigators have shown VLDL secretion to increase (Topping and Mayes, 1972; Laker and Mayes, 1984) have no effect (Whodside and Heimberg, 1976; Beynen et al., 1981; Beynen and Geelen, 1982; Haagsman and Van Golde, 1984) or to decrease (Nikkila, 1984; Durrington et al., 1982; Patsch, 1983 ; Pullinger and Gibbons, 1985; Mangiapane and Brindley, 1986). In the cases where either no effect or a decrease was observed, the investigators utilized rat hepatocytes, whereas the increased VLDL secretion was observed with perfused rat livers and in one case, a long term insulin treatment 12 vivo (Steiner et al., 1984). Chick 26 hepatocyte cultures require insulin for secretion (Tarlow et al., 1977) as removal of insulin results in a parallel decline in acetyl CoA carboxylase activity and VLDL synthesis. This effect could be reversed by preincubating the cells for 2-3 days with insulin. Caro and Amatruda,(1982) used isolated hepatocytes from fasted rats and found it necessary to preincubate the cells for 20 hours with insulin before they observed a glucose stimulation of acetyl CoA carboxylase activity. An increase in acetyl CoA carboxylase should correlate with an increase in hepatic fatty acid synthesis which in turn would provide substrate for VLDL production. Addition of glucagon to the media blocks VLDL secretion presumably by modulating the degree of phosphorylation of acetyl CoA carboxylase (Tarlow et al., 1977). Therefore, it appears that in the short term regulation of lipoprotein metabolism is related to phosphorylation-dephosphorylation mechanisms on key lipogenic enzymes, whereas long term effects may be due to induction of enzyme synthesis (Forte, 1984). Insulin action on key lipogenic enzymes offers a reasonable explanation as to why increases in VLDL secretion might be expected. The observations of others that have observed decreases in VLDL secretion are not as easily explained. Mangiapane and Brindley,(l986) found that preincubation of isolated rat hepatocytes for 19 hours with insulin decreased the total amount of TC in cells and medium. This was not observed when insulin was added at the beginning of the incubation. In addition, the relative activity of 27 phosphatidate phosphohydrolase was not changed in the presence of insulin compared to control. The only explanation offered for these observations and the discrepancies in the literature were related to the experimental system used and availability of substrate and their alternative metabolic routes. Further work needs to be done to better characterize these contrasting observations on effects of insulin. Glucagon, on the other hand, as previously mentioned, and cAMP analogues inhibit fatty acid synthesis (Beynen et al., 1979), phosphatidylcholine biosynthesis (Pelech and Vance, 1984), and synthesis and secretion of TG by VLDL in rat hepatocytes (Beynen et al., 1981; Kempen, 1980; Haagsmmn and VanGolde, 1984) and in perfused rat livers (Heimberg et al., 1969). The cAMP mediated inhibition of fatty acid synthesis and phosphatidylcholine are presumably regulated by phosphorylation-dephosphorylation mechanisms. The effect on actual VLDL secretion may be related to direct effects on VLDL synthesis or secretion or to the effects on substrates (i.e. fatty acid and PL availability) (Vance and Vance, 1985). Mooney and Lane,(1981) using a chick hepatocyte system found that cAMP caused mobilization of TG rich vesicles (TGRV), earlier referred to as floating fat. An increase in lipolysis was observed and attributed to some form of cAMP activated TG lipase. Although the existence of such an enzyme has been shown in adipose tissue (Shapiro, 1977) it has not been established in liver. A lysosomal 28 hepatic TG lipase in rat hepatocytes has been identified by Debeer et aL,(1979 and 1982) and may be indirectly regulated by glucagon. Glucagon induced the formation of autophagosomes, membrane bound vacuoles which undergo fusion with lysosomes (Debeer et al., 1982). The cAMP role may be to facilitate the presentation of the TGRV into direct contact with lysosomal lipase (Mooney and Lane, 1981). Additionally, the cAMP mediation favors production of oxidative products such as acetoacetate and betahydroxy- butyrate over VLDL secretion (Mooney and Lane, 1981). Sex hormones are also believed to regulate VLDL secretion in normal animals. Perfused livers of female rats secreted more VLDL-TG at given perfusate fatty acid levels than did males (Soler-Argilaga and Heimberg, 1976; Watkins et al., 1972). Moreover, pregnant rats secreted more TG than nonpregnant rats (Wasfi et al., 1980). This increase may be a result of several hormones acting in concert during gestation. Plasma concentration of estrogens (Costrini and Kalkhoff, 1971), insulin to glucagon ratio (Saudek et al., 1975) and glucocorticosteroids (Pekkarinen et al., 1962) are known to increase during pregnancy. Estrogen (given as ethinyl estradiol) has been shown to stimulate TG esterification, VLDL secretion and reduce ketogenesis in perfused livers and hepatocytes of female rats (Weinstein et al., 1979, 1986). High doses of ethinyl estradiol resulted in an increase in the molar ratio of CE to T0 from 8 to 35 for pair fed controls vs treated rats. This may have been due 29 to stimulation of the acylcholesterol acyl transferase (ACAT) which converts cholesterol to its ester form (Weinstein et al., 1986). In hens, at the onset of the laying period, increases in estrogen stimulate hepatic VLDL production (Kudzma et al., 1975 and Schjeide et al., 1963). An estrogen induced synthesis of VLDL apoprotein B has also been described in birds (Chan et al., 1974; Miller and Lane, 1984). Incorporation of [3H]-leucine into apolipoproteins B and C was increased in chick hepatocytes pretreated with estrogen (Tarlow et al., 1977). These studies suggest a role of estrogen in regulation of lipid and protein synthesis in hepatic.VLDL production. Glucocorticoids have also been shown to stimulate hepatic TG synthesis in rats and in fact, may lead to production of fatty liver (Hill and Droke, 1963; Ozegovic et al., 1975). Injections of several corticosteroid analogues over several days increase hepatic secretion of VLDL (Reaven et al., 1974; Krausz et al., 1981; Cole et al., 1982). Direct addition of dexamethasone to a liver perfusion increased secretion at very high hormone concentration only (Cole et al., 1982). Preincubation of isolated hepatocytes with luM.dexamethasone for 19 hours followed by further incubation for 23 hours resulted in 2 to 4 fold increases in TG secretion as VLDL (Mangiapane and Brindley, 1986). These researchers also observed an increase in the relative activity of phosphatidate phosphohydrolase,some 11 fold greater than control, consistent with other reports 30 (Pittner et al., 1985). The response to dexamethasone may involve long term.regulation such as enzyme synthesis of specific enzymes involved in substrate formation as indicated by the length of time required for a response. One other hormone which appears to affect VLDL metabolism is thyroid hormone. In liver perfusion studies of rats made hyperthyroidemic, VLDL secretion was decreased 502 compared to euthyroid rats (Olubadewo et al., 1983). This effect was recently suggested to be due to a triiodothyronine (T3) induced decrease of glycerol-3-phosphate as glycerol addition restored VLDL secretion to levels observed in the euthyroid status (Olubadewo and Heimberg, 1985). It was also noted that the T3 treated rats diverted more of the substrate fatty acid to ketone body production (Olubadewo et al., 1983; Olubadewo and Heimberg, 1985). Other studies have been carried out to evaluate VLDL secretion in hypothyroid rats (Calandra and Tarugi, 1984). A reduction in hepatic fatty acid synthesis "de novo" has been demonstrated in yiyg_and ig_ giggg (Dayton et a1, 1960; Boyd, 1961). More recent studies have confirmed these findings and also shown decreases in hepatic cholesterol, CE, fatty acid and TG synthesis (602 to 802) as well as decreased secretion of TG into the medium (Tarugi et al., 1981). Reduced activity of fatty acid synthetase (Volpe and Kishimoto, 1972) and acetyl CoA carboxylase (Volpe and Vagelos, 1976) have been observed following thyroidectomy. The decrease in these key lipogenic enzymes is consistent with the reduced lipogenesis. Some 31 reports have indicated that thyroid hormone regulation occurs at the site of mRNA synthesis for specific enzymes (Towle et al., 1980, 1981; Miksicek and Towle, 1982). A reduction in apoprotein incorporation into VLDL also occurs as indicated by studies of Calandra and Tarugi (1982). These researchers found a 402 decrease in the amount of [l4-C] amino acid incorporated into VLDL secreted by rats made hypothyroid. Thyroid hormone appears to regulate hepatic synthesis of both the protein and lipid constituents of the VLDL. Characteristics of Circulating Lipoproteins This portion of the review will attempt to construct a general review of plasma lipoprotein metabolism in bovine species focusing particularly on hepatic VLDL contribution to this metabolic scheme. A variety of methods have been used to separate lipoproteins into their respective classes. These include density ultracentrifugation (Havel et al., 1955; Grummer et al., 1983), gel filtration (Ferreri and Gleockler, 1979; Grummer et al., 1983), sulfated polysaccharide precipitation (Burnstein and Samaille, 1957), heparin sepharose chromatography (Cordle et al., 1985), high performance liquid chromatography (Carroll and Rudel, 1983) and immunoprecipitation techniques (Salmon et al., 1984) The general classification for lipoproteins based on density ultracentrifugation and electrophoretic mobility is as follows: 1) chylomicrons (CM) in a density range of less than .95 g/ml which do not migrate from the origin; 2) VLDL-.91- 1.006 g/ml with pre-beta mobility; 3) low density lipoprotein 32 (LDL) l.019-1.063 g/ml with beta mobility; and 4) high density lipoprotein (HDL) -1.063-1.21 g/ml with alpha mobility (Kris-Etherton and Etherton, 1982). A further sub- classification of HDL into HDLl (1.063-1.090 g/ml), HDL2 (1.090-1.12 g/ml) and HDL3 (1.12-1.21 g/ml) also exists in human lipoprotein classification, as well as an intermediate density lipoprotein (IDL) in the range of (1.006-1,019 g/ml) (Puppione, 1978). A similar IDL has been characterized in bovine serum by Puppione et al., (1982). Interpretation of the literature on separation of bovine lipoproteins is difficult due to the variety of methods used among investigators, differences between lactating and non- lactating animals (Raphael et al., 1973, a,b) and temperature effects during lipoprotein isolation (if carried out below the range of 25°-38°C) (Puppione, 1983). Attempts to characterize bovine lipoproteins using density ultracentrifugation have met with variable results, especially in the density range l.006-1.063 g/ml. Stead and Welch,(1976) subfractionated bovine LDL using ultra- centrifugation into LDLl, (l.019-1.039 g/ml) and LDL2 (1.039-1.060 g/ml). The LDLl exhibited alpha migration while the LDL2 exhibited beta on disc polyacrylamdde gel electrophoresis (PAGE). This led them to suggest that the LDLl class was more closely related to HDL, while LDL2 was similar to VLDL. Others have also separated the LDL fraction into two distinct classes (Dryden et al., 1971; Raphael et al., 1973a). However, Raphael et al., (l973a),found that the LDLl 33 fraction (1.006-1.039 g/ml) exhibited beta mobility while the LDL2 fraction (l.039-1.063 g/ml)exhibited alpha mobility. They called this fraction LDLl, as it more closely resembled HDL. In addition, the amount of overlap between alpha and beta migration was dependent on the physiological state of the cow (lactating and non-lactating). Other workers using gel filtration to separate the LDL fraction confirmed these results (Ferreri and Gleockler, 1979). Further studies of Grummer et al., (1983)_separated this HDLl fraction (1.039- 1.063 g/ml) into two peaks. Through the use of ultra- centrifugation followed by gel filtration and confirmed by immunoelectrophoresis and double immunodiffusion techniques, they showed that the first peak corresponded to LDL and the second to HDL. It would appear, therefore, that this is not a distinct classification of lipoproteins. Table 1. (taken from.Puppione, 1983). Composition of the Majgr Bovine Lipoprotein Classes. Percent by Weight ___c1ass 3.29. 21:3. 2.1: 19 9.9. Chylmmicron 3 2 4 87 4 VLDL 8 5 7 74 7 B-LDL 24.0 39.3 29.2 1 8 5.7 a-LDL 21.8 38.6 32.2 -- 7.5 HDL-Lac. Cow 33.4 33.3 29.5 -- 3.9 " Steer 37.5 37.3 22.4 -- 2.8 " Calf 45.1 29.0 20.6 -- 2.7 " Fetus 50.3 22.9 23.8 -- 3.0 Protein (Pro), cholesterol esters (CE), phospholipid (PL) triglyceride (TG) and unesterified cholesterol (UC). 34 As indicated by Table 1, protein, CE and PL make up most of the LDL and HDL fraction, while TC is the major fraction of VLDL and CM. Studies by Raphael et al., (1973a) demonstrated that bovine HDL was the major lipid bearing class of plasma lipoprotein (572 to 762), while VLDL accounted for less than 52. Others have demonstrated that the VLDL concentration is low as well (Christie, 1979). .In the lactating cow, (Raphael et al., 1973a) has characterized the changes in lipoprotein fractions that occur throughout lactation and gestation. These researchers noted that VLDL concentration was low (5-7 mg/dl) from 0-33 weeks postpartum and increased to a level of 14 mg/dl at 0-15 weeks prepartum. They further observed that LDL, HDLl and HDL fractions all were low from 0-4 weeks post partum (10 mg, 70 mg and 223 mg/dl, respectively) and increased at 5-10 weeks to a level of 35 mg, 162 mg and 327 mg/dl, respectively, corresponding to peak milk production. Measured at 16-33 weeks prepartum, there were no significant changes in the LDL or HDLl fraction, but the HDL fraction decreased to 260 mg/dl. Finally, all three fractions decreased during the dry period to a level of 16 mg, 54 mg, and 190 mg/dl, respectively. It was also noted that the lipid composition throughout was fairly stable. It was hypothesized that the low VLDL level in early lactation was due to increased lipolysis and uptake of TG by LPL. In fact, LPL activity in the mammary gland has been shown to increase dramatically upon initiation of lactation while it decreases dramatically in adipose 35 concomittant with an increase in adipose lipolysis (Chilliard and Robelin, 1985). Additionally, as mentioned previously, the half life of TC in the lactating cow has been shown to be very rapid by Palmquist and Mattos, (1978) (1-2 minutes) and Glascock and Welch, (1974) (about 4-5 minutes.) Therefore, the increases in LDL, HDL1 and HDL may reflect the increase in VLDL metabolism. It would be interesting to follow plasma LCAT activity throughout lactation to see if there is a corresponding pattern. Another observation on bovine lipoprotein metabolism is the formation of rather large alpha lipoproteins. Over 502 of bovine alpha HDL in the lactating cow has been found in the density interval of 1.063 to 1.090 g/ml (Puppione et al., 1982). In addition, the mean diameter of bovine HDL appears to be larger than in other mammals (Forte, et al., 1979, 1981). There may be a number of reasons for this observation. Plasma LCAT may mediate the conversion of small alpla lipoproteins to the large form (Glomset and Norum, 1973). This could happen in the lactating animal when there is an increase in VLDL lipolysis in turn shedding substrate for LCAT. A second possibility is that the formation of the large lipoprotein reduces the total number of lipoproteins needed to transport the cholesterol esters (important especially if apo A-I were limiting) (Puppione, 1983). Thirdly, it may be a metabolic response to increase the resevoir of apo C by increasing the surface area of the lipoprotein and in turn providing apo C for chylomicron and VLDL. Havel et al., (1973) has proposed this mechanism for 36 other mammaliam systems. Role of Apolipoproteins The predominant apoprotein in bovine plasma has a molecular weight of approximately 28,000 (Lim and Scanu, 1976), and is analagous to the apo A-I in human plasma. Apo A-I synthesis occurs in the liver (Lin-Su et al., 1981) and intestine (Gordon et al., 1982) of the rat. It has also been found in the livers of humans, pigs, chickens and guinea pigs (Marsh, 1984). Wu and Windemueller, (1979) estimated hepatic and intestinal synthesis of apo A-I by intraportal or intraduodenal injection of labelled leucine and estimated that about 442 was of hepatic origin. Apo A-I has been shown to be an activator of lecithin cholesterol acyltransferase (LCAT) enzyme in plasma (Fielding et al., 1972; Noble et al., 1972). This enzyme catalyzes the transfer of the beta fatty acid from lecithin and transesterfies it to cholesterol to form CE. About 802 of the protein associated with HDL is Apo A-I and the rest is associated with a group of five to seven low molecular weight (7,000 to 10,000) apoproteins (Puppione, 1983). These are believed to correspond to human apo A-II, apo C-II, apo C-III-O, apo C-III-l and apo C-III-2 (Patterson and Jones, 1980). Apo A-II is an important constituent of HDL3 in humans. It can stimulate hepatic lipase (Jahn et al., 1983), but it may not be an essential co-factor. Apo C-II, an essential co-factor for lipoprotein.lipase (LPL) appears to be synthesized only in the liver, along with the other apo 37 C-III forms (Marsh, 1984). Apo C—III has been shown to inhibit hepatic uptake of VLDL and chylomicron remnants (Windler et al., 1980; Shelburne et al., 1980) and of the original TG rich particles (Windler and Havel, 1985). Low amounts of what may be analagous to apo A-IV and apo A—V have also been found on bovine alpha HDL in densities greater than 1.10 g/ml (Puppione et al., 1982). Apo A—IV is made by both intestine and liver of rats and is one of the chylomicron apoproteins (wu and Windmueller, 1979). The function of apo A-IV remains to be determined. Apoproteins analagous to apo E have also been found in trace amounts of bovine alpha HDL (Tall et al., 1981). Apo E plays a major role in hepatic receptor binding of lipoprotein remnants (Hui et al., 1984). The major site of synthesis of apo-E is the liver (Marsh, 1983) and small amounts have been found in kidney (Blue et al., 1983). Finally, there appears to be two forms of apo B in bovine plasma, although their molecular weights and sites of synthesis have not been determined (Puppione, 1983). As mentioned earlier in this review, human and rat apo B has been shown to exist in at least two distinct forms, a high molecular weight form (apo B-100) and a low molecular weight form (apo B-48) (Kane, 1983). In adult rats, hepatic VLDL contain both apo B-48 and apo B-100 (wu and Windmueller, 1981; Sparks et al., 1981) while hepatic VLDL from humans (Hui et al., 1984), guinea pigs (Guo et al., 1982) and suckling rats (Imaizumi et al., 1985), contain only apo B-100. The apo B-48 is predominant in lymph chylomicrons of 38 humans and rats (Kane et al., 1980; Krishnaiah et al., 1980). The significance of these two forms of apo-B in relation to lipoprotein metabolism will be discussed in the next section. Finally, a recent, more comprehensive review of the apolipoproteins is available (Mahley et al., 1984). Lipoprotein Catabolism Controversy has existed in the past as to whether bovine species do form chylomicrons for transport of absorbed dietary fat. Gage and Fish,(1924) were the first to report chylomicrons in the blood of a dairy cow on a normal diet of bran and hay. More recently, evidence has accrued for the existence of the chylomicron-size particles (Ferreri and Elbein, 1982). The half life of chylomicron in lactating cows is very short (1-l.5 minutes) indicating that these particles are metabolized very rapidly (Palmquist and Mattos, 1978). Studies with non-ruminants indicate that chylomicron particles, containing at least two ap0proteins (apo B-48 and apo A-I) are secreted into the lacteals where they acquire apo C-II from HDL and probably shed A-I in association with cholesterol and PL (Imaizumi et al., 1976). Once they acquire the apo C-II, they interact with LPL in extrahepatic tissues and form chylomicron remnants (Havel et al., 1980). These remnants still contain apo B-48 and are rapidly removed by the liver without conversion to LDL (Van't Hooft et al., 1982; Floren, 1984). These chylomicron remnant particles also contain apo E, which has been shown to be a necessary constituent for effective removal by a specific apo E hepatic 39 receptor (Hui et al., 1984) The metabolic fate of VLDL is similar to that of the chylomicron. Hepatic VLDL are secreted with apo B and acquire apo C-II from the HDL in plasma (Puppione, 1978). The VLDL then interact with LPL in extrahepatic tissues and form a VLDL remnant. In humans, this VLDL-apo B-100 particle has two possible fates: 1) it may be removed by a second hepatic receptor (LDL apo B-E), distinct from the apo E receptor previously mentioned (Hui et al., 1984), or 2) it may be converted to LDL (Havel et al., 1980), which can then be cleared by the hepatic LDL apo B-E receptor (Hui et al., 1984), or similar receptors located in other tissues, e.g. spleen or adrenal (Kovanen et al., 1979). The liver, however, is quantitatively the most important organ involved in LDL catabolism (Attie et al., 1982). Plasma clearance of hepatic VLDL-TG (in xiyg) has been widely studied through the use of radioisotopes in man (Havel et al., 1970; Shames et al., 1970; Quarfordt et al., 1970; Havel and Kane, 1975; Zech et al., 1979), rats (Baker and Schotz, 1964, 1967), rabbits (Havel et al., 1962), dogs (Havel and Goldfien, 1961), and chickens (Kudzma et al., 1975; Bacon et al., 1978). Studies by Grundy and Mok, (1976) 1.h-1) in human utilized a duodenal lipid infusion (200 mg.kg' subjects that were normal or hyperlipidemic and measured chylomicron clearance rates. Blood was sampled over a five hour period during steady state. Clearance rates in normal subjects were rapid (t 1/2 = 4.5 i 3 min) whereas those in 40 hyperlipidemic subjects were more prolonged (t 1/2 = 23 i 5.5 mdn). A defect in removal of chylomicron was discounted by the observation that a reduction in endogenous TG by caloric restriction resulted in clearance rates returning to normal. Therefore, the hyperlipemia was apparently due to increases in hepatic VLDL production, which in turn may have caused competition for removal between the endogenous (hepatic VLDL) and exogenous (dietary chylomicrons) particles resulting in the longer half life observed in hyperlipidemic subjects. Differences in clearance rate related to particle size have been observed in the rat (Quarfordt and Goodman, 1966) and in man comparing turnover of intravenous fat emulsions and endogenous TG (Rossner et al., 1974). Other studies with human subjects have demonstrated half lives for endogenous T6 to be on the order of one hour in normotriglyceridemic and three to five hours in type IV hypertriglyceridemic (increased hepatic production of VLDL) human subjects (Quarfordt et al., 1970). Similar values have been reported for chickens («~-l hour Kudzma. et al., 1975), rats (a~2.8 hour-Baker and Schotz, 1967), rabbits (a-1.5 hour-Havel et al., 1962), and dogs (*-1.8 hour-Havel and Goldfein, 1961). .Comparable values for endogenous TG have not been determined in ruminants, although several studies have observed very rapid half lives on the order of one to five minutes for exogenous TC in sheep (Bergman et al., 1971) and lactating cows (Palmquist and Mattos, 1978) as mentioned previously. It does appear that endogenous-TC is removed much less efficiently than exogenous 41 TC and removal may be related to the type of lipoprotein that carries the TG (Grundy and Mok, 1976). Perfusion studies (Hamilton et al., 1976) have suggested at least one possible origin for HDL. These investigators perfused rat liver with LCAT enzyme inhibitor and isolated perfusate HDL containing unesterified cholesterol, PL and apoprotein (primarily apo E). _Following a negative stain of electron micrographs, these HDL particles appear as discs. Normal plasma HDL are more spherical (Forte and Nichols, 1972; Puppione et al., 1971). Similar discoidal particles have also been detected in intestinal lymph secretions (Kris-Etherton and Etherton, 1982) and in plasma of individuals with an LCAT deficiency (Forte and Nichols, 1972; Torsvik, 1972). These disc shaped particles appear to be the preferred substrate for LCAT (Puppione, 1978). More recently Eisenberg, (1979) has suggested that most of the HDL may be formed from.the shedding of the surface lipid constituents of VLDL and chylomicron. The TC rich particle reacts with LPL releasing cholesterol, PL and apoprotein A-l, which combine and form substrate for plasma LCAT, resulting in formation of HDL particles. This has been demonstrated 32, 31552 (Eisenberg and Olivercrona, 1979; Patsch et al., 1978) and ip vivo (Tall et al., 1982). 42 Contribution to Milk Fat Having discussed the general lipoprotein metabolic scheme, the next question to address (alluded to in the beginning of this review) is hepatic VLDL contribution to the mammary gland. The physicochemical properties of lipoproteins secreted by bovine liver are unknown at the present time. It is presumed that in early lactation, adipose tissue lipolysis provides NEFA substrate for hepatic VLDL production (Puppione, 1983). Direct net uptake of NEFA by the mammary gland has been shown to be small and variable in most cases and not likely to contribute much to milk fat TG (Glascock et al., 1966; Bishop et al., 1969; Annison et al., 1967b; Barry et al.,l963). Studies by Annison et al., (1967b) detected considerable transfer of labelled fatty acid into milk fat of lactating goats, although net NEFA arteriovenous differences across the mammary gland were small. In addition, there was a marked decline of NEFA specific activity across the mammary gland. The possibility that hydrolysis of TG contributed NEFA which diluted the specific activitijas suggested. One study has reported significant arteriovenous differences in the lactating COW’When arterial NEFA concentration exceeded 300 qu/L (Kronfeld, 1965). The net arteriovenous differences reported in this study ranged from 40 to 200 qu/L (one value of 400 qu/L). High producing cows in early lactation have increased concentrations of plasma NEFA due to rapid mobilization of body stores in response to negative energy balance. Therefore, direct NEFA 43 uptake by the mammary gland may be occurring at this stage (Kronfeld, 1965). A number of studies have reported mammary uptake of TG (Glascock et al., 1966; Bishop et al., 1969; Annison et al., 1967m and more specifically VLDL-TC (Barry et al., 1963; Palmquist, 1976), but the endogenous (via hepatic VLDL) vs exogenous (dietary) contribution was not determined. Palmquist and Mattos, (1978) attempted to answer this by following the transfer into milk fat of: 1) labelled fatty acid dosed ruminally; or 2) by preparing VLDL and chylomicrons from calf lymph (Mattos and Palmquist, 1977) and dosing these iv. Data derived from the radioactive decay curve in milk fat by curve peeling (Shipley and Clark, 1972) suggested that the exogenous contribution was between 852 - 902, while the endogenous was 102 - 152. These results were similar to a previous study by Palmquist and Conrad, (1971) when results were recalculated from the zero time estimates. These data do suggest that dietary lipoproteins are the predominant precursor for milk fat. None of these studies differentiated between production level or stage of lactation so that the early part of lactation has not been evaluated in this respect and may in fact differ (Palmquist and Mattos,l978).T0 what extent mobilized NEFA are in fact substrate for hepatic VLDL synthesis and secretion in early lactation in ruminant species remains to be determined. RUMINANT HEPATIC TRIACYLGLYCEROL SECRETION g VITRO 44 MATERIALS AND METHODS 111 VITRO This section describes the procedures used to measure secretion of triacylglycerol from liver slices of various species. Source of Tissue Cattle, sheep and pig livers were obtained from the Michigan State University Meats Laboratory. Cattle were stunned with a captive bolt pistol, rapidly hung and exsanguinated. Sheep and pigs were stunned by the use of humane electrical stun and exsanguinated. Initially, liver samples were generally removed within 15 to 25 minutes of exsanguination and incubating by 30 to 35 minutes. Later the time from slaughter to incubation was rapidly reduced to 8 to 10 minutes. Bovine samples were generally obtained from beef breeds (steers and heifers) and an occasional Holstein (steer). Ovine samples were generally from crossbred lambs or spent ewes from the M.S.U. sheep barns. Pig samples were from boars on experiment at the M.S.U. swine facility. All animals had been on feed within 4 to 14 hours of the time of slaughter. Beef breed animals were fed typical growing or finishing rations (corn:corn silage) fed at M.S.U. beef research facility. One Holstein bull calf (2 weeks of age) fed a primarily milk diet was also obtained from the M.S.U. dairy facilities. Lambs were fed typical growing finishing 45 46 type rations, while ewes were on pasture. One neonate crossbred lamb was also obtained from the M.S.U. sheep barns within 2 hours of birth (separated from dam immediately after birth). The boars were control animals on an experiment and had been fed typical boar rations of corn:soybean meal. Sprague-Dawley rats and New Zealand White rabbits were obtained from the M.S.U. Biochemistry animal care facility and had been fed typical Purina rat or rabbit chow. Some of the rats were at the end of the third week of lactation and others were medium-sized males and females (200-300 g B.W.). A litter of rats was also raised in Anthony Hall rat room to about one year of age (500-600 gms. B.W.). The rabbits were male or female and weighed 3.5 to 4.0 kg. All these animals were on feed prior to decapitation. Single Comb-White Leghorn hens were obtained through the M.S.U. poultry facility and were slaughtered in the M.S.U. poultry processing facility in the basement of Anthony Hall. The hens, in various stages of lay, had been maintained with a typical corn:soybean meal layer type diet until slaughter. Fish were obtained from the M.S.U. fisheries research facility on Kalamazoo Street. They were maintained with Purina Adult Fish Chow in the trout and salmon tanks at 15°-20°C. The fish were transported to the laboratory alive and decapitated immediately, following stunning. The fish were either Rainbow or Brown trout about 800-1000 g in weight. Finally, a guinea pig was obtained from the Physiology 47 Department in Giltner Hall. It was maintained with typical guinea pig chow and had not been on experiment. Tissue Preparation Liver samples obtained at the meats laboratory or poultry processing facility were transported to the incubation site within 2 minutes. All other samples were obtained at the incubation site. Liver tissue was cut into strips (approximately 1 cm x 1 cm x 2 cm) and placed into ice cold Minimal Essential Media (MEM), pH 7.4 without L-glutamine; with 25 mM Hepes buffer and with Earle‘s salts obtained from GIBCO Laboratories, Grand Island, New York (See Appendix Table 1: Eagle, 1959). Tissue blocks were trimmed to remove capsule and nonparenchymal tissue and were sliced with a Stadie-Riggs microtome, grooved to produce slices about .5 mm thick. Initially these operations were all conducted on ice. Liver slices were placed in ice cold media prior to weighing on a Mettler digital balance (Model A30: Mettler Instrument Corporation, Hightown, New Jersey). Liver slices, weighing between 40-80 mg each, were incubating within 8 to 35 minutes frmm the time of exsanguination. Excess liver was stored at -60°C for lipid extraction to determine percent lipid in liver. Triacylglycerol Esterification and Secretion Studies Solutions of 3 H-oleic acid and 1-14 C palmitic acid (in toluene, New England Nuclear, Boston, Mass.) were stored as purchased at -20°C. Solutions of each were made up in toluene and stored at 4°C for easy access. All other 48 chemicals, hormones and antibiotics were from Sigma. Media Preparation A stock solution of 1.25 mM bovine serum albumin (BSA), essentially fatty acid and globulin free (Sigma, St. Louis, Missouri) was prepared by dissolving the BSA in 10 mM KHZPOA, pH 7.4 and warming while stirring on a hot plate (Sybron/Thermolyne Model Nuovant heating stir plate, Dubuque, Iowa) at setting 3. Too much heat causes the BSA to gel, therefore caution should be exercised. Following dissolution of the BSA, it was filtered and the pH was checked. Cold substrate fatty acid (5 mM) was added as either palmitic acid (Na+ salt), oleic acid or a 1:1 combination. The solution was again gently warmed and stirred for 3 to 4 hours. The final fatty acid:BSA ratio was 4. The radio-labelled fatty acid was prepared by evaporating sufficient label under N2 to obtain about 1.7 x 106 dpm/flask (2500-3000 dpm/nmole fatty acid) in the final media preparation. An aliquot of unlabelled BSA-fatty acid solution was added to the labelled material and dissolved. The final solution was determined to be dissolved (after 2 to 3 hours) by scintillation counting. The substrate was added to the desired amount of MEM along with appropriate amounts of Penicillin G (PEN-NA; .05 mg/ml) and Streptomycin Sulfate (.05 mg/ml). Finally, 3 ml of the final media mixture (.2 mM fatty acid) was pipetted into 25 m1 erlenmeyer flasks and stoppered. Media was prepared the night before the experiment and stored at 4°C. Media was prepared fresh for each experiment. 49 Incubation Procedure The liver slices were blotted on tissue paper, placed in the incubation flasks, weighed and gassed with a 95:5 mixture (02:002) for 20 seconds. The flasks were sealed with rubber stoppers and placed in a 37°C water bath (20°C for fish liver; Rogie and Skinner, 1985) in a Dubnoff Metabolic Shaking Incubator (Precision Scientific, Chicago, Illinois) oscillating at 60 cycles/minute. All treatments were performed in quadruplicate. Flasks were gassed 30 seconds every 1.5 hour throughout the period of the experiment, generally lasting up to 8 hours. Incubations were terminated by placing the flasks on ice and separating the media from the tissue. Blank or zero time samples were placed on ice immediately while other samples were terminated at 4 hours, 6 hours, 8 hours or up to 12 hours, depending on the experiment. Tissue and media samples were stored frozen (-60°C) until eXtraCted. Sample Extraction and Separation Tissue samples were extracted according to the methods of Hara and Radin,(1978). Butylated hydroxy toluene (BHT) (100 ul of 12 solution) was added to the extracts prior to evaporation under N2 and transfer to 12 x 75 mm test tubes. Addition of BHT to lipid extracts inhibited formation of a varnish-like substance if extracts were exposed to air. Recovery of lipid in tissue extracts was 902 to 952. Media 'samples were extracted according to methods of Hara and Radin,(1978) with some adaptations (See Appendix 2 for details). 50 The extraction method was maintained at neutral pH to minimize the partition of NEFA which increased contamination of the TG spot following thin layer chromatography. The extraction then maximized partition of the less polar components (i.e. TG, CE) while minimizing that of NEFA. The samples were evaporated under N2 following addition of BHT and were transferred to 12 x 75 mm test tubes. Aliquots of sample were spotted on thin layer plates (Uniplate; Analtech Inc., Newark, Delaware) coated with silica gel HL (No. 46011) following activation in a 100°C oven for 1 hour. A Sigma lipid standard (No. 178-3) containing tripalmitin, dipalmitin and monopalmitin plus added palmitic acid was co-chromatographed with the samples. Plates were developed in hexane:diethyl ether:acetic acid (50:50:4) for about 45 minutes in a developing chamber. Following removal, the plates were dried and sprayed with .22 2 , 7 dichlorofluorescein lipid stain (Sigma No. D 6132) in ethanol. Spots corresponding to TG standard spots were scraped into scintillation vials and following addition of scintillation fluid (Safety Solve Research Products, Int. Corporation, Mt. Prospect, Illinois) were counted in a TM Analytic Model No. 6872 scintillation counter (Elk Grove, Illinois). 51 Calculations Fatty acid esterified into hepatic TG was calculated as follows: Total nmoles = (dpm in sample - dpm in blank) x pmoles/dpm divided by mg spotted x mg tissue wt. divided by 1000. pmoles fatty acid esterified per mg = total nmoles x 1000 divided by mg tissue. Fatty acid secreted into the media as TG was calculated in a similar manner varying the dilution factors. Total nmoles = (dpm in sample-dpm in blank) x pmoles/dpm x .001 x dilution factor (3/2) x 4. pmoles fatty acid secreted as TG per mg = total nmoles x 100 divided by mg tissue. Total values esterified or secreted were representative of complete incubation periods (eg. 4, 6, 8, 10 or 12 hours). Statistical Analysis Data were analyzed as factorials or in some cases using an orthogonal test for linearity. Appropriate f-tests or t-tests were used to test significance of the response (p4<.05). Individual liver slices were considered as independent variables when one animal was used per experiment, However, when comparing species differences individual means (from n =4 replicates) of each animal as an experimental unit were compared. RESULTS IN VITRO Assay Development The results of preliminary studies with a female rat are shown in Table 2. After subtracting the blank or zero time Samples from the 4, 6, and 8 hour samples, a significant increase (p'<.05) in both tissue esterification and secretion of fatty acid as TG into the media was observed. Table 2. Esterification and Secretion of Fatty Acid as TC in Liver Slices from a Female Nonpregnant Rat. h n media n tissue -------- pmoles/mg - - - - - - - - - - 4 2 9.4 i 4 4 127 i 31 6 2 47.1 i 12 2 170 i 63 8 2 60.4 i 9 4 180 i 17 Values are means i S.E.M. for n replicates of liver slices, 111 i 45 mg each. Incubation procedures were as described in Methods. Media contained .2mM palmitate and flasks were gassed every 45 minutes with 95:5 02:C02. Final specific activity was .5478 pmoles/cpm. Zero times subtracted out. Results from two preliminary ewe studies are shown in Table 3. In Ewe I secretion rates were low but did increase with time over the 4, 6, and 8 hour periods. The negative value observed at 4 hours in ewe I and 4 and 6 hours in ewe II resulted because the means were lower than the 0 hour blanks. An explanation of this contamination problem and a discussion 52 53 of what was done to minimize this follows in the discussion section. The reduced secretion rates in the ewe livers compared to the rat made minimizing the contamination problem even more critical. Esterification rates, however, were similar between ewes I and II and increased linearly with time (p< .01 and p<.025, respectively) suggesting that the tissues were viable. Results in steers I and II (Table 4) are similar to those observed in sheep. Addition of propionate, a possible limiting factor to normal ruminant hepatic metabolism did not appear to stimulate secretion of fatty acid as TC in the media. Tissue esterification rates were linear (p«<.01) and slightly higher than those observed in ewes (Table 3). The tissue appears to have been viable throughout the course of the incubation. Further attempts to manipulate the media in order to enhance secretion of TG are shown in Tables 5 and 7. Both the addition of serum (102) to the media and type of media (Krebs Ringer (KRB) vs Minimal Essential Media (MEM)) did not appear to enhance secretion of TG from steer liver slices (Table 5). However, esterification rates were quadrupled in the MEM vs the RER media at the 6 hour time period. 54 Table 3. Esterification and Secretion of Fatty Acid as T6 in Ovine Liver Slices (Ewes I and II). media tissue h n I n 11 n I n 11 --------- pmoles/mg - - - - - - - - - - - - - — 4 2 -1.1 i 1 4 -7.3 i .75 4 262 i 31 4 235 i 20 6 2 4.9 i .85 4 -4.4 i 2.7 4 406 i 38 4 356 i 56 8 2 7.9 i .95 4 .4 i 2.4 4 499 i 42 4 459 i 30 Values are means i S.E.M. for n replicates of liver slices (x = 91 i 38 mg - Ewe I; 49 i 8 mg - Ewe II). Incubation procedures were as described in Methods. Media contained .2mM palmitate and flasks were gassed every 30 minutes with 95:5 0 :C02. Final specific activity was .13106 pmoles/cpm. Zero times subtracted out. Table 4. Effect of Added Propionate (1.0 mM) on Esterification and Secretion of Fatty Acid as TC in Steer Liver Slices (Steers I and II). media tissue h n I II n I II --------- pmoles/mg - - - - - - - - - - - - - - - 4 4 -l.5 i 3 l i .7 4 383 i 7.5 467 i 26 6 4 _10 i 4.5 2 i .75 4 561 i 44 772 t 264 8 4 . 8 i 4.0 7 i .9 4 631 i 69 880 i 123 Values are means i S.E.M. for n replicates of liver slices (x = 132 i 32 mg - Steer I; 86 i 18 mg - Steer II). Incubations were as described in Methods. Media contained .2mM palmitate and flasks were gassed every 30 minutes with 95:5 02:CO . Final specific activity was .0677 pmoles/cpm (Steer I) and .0449 pmoles/cpm (Steer II). Zero times subtracted out. 55 .uso umuomuunsm moawu ouoN .an\moHoEm maomm. mm? hufl>wuom owwfioomm Madam .NoonwoAmummv nuaB mouacwa om mum>o powwow ouoz mxmmam .NOH um poops mm3.anuom can oumcoaaoun 29H .oumuwsamm ZEN. poawmuaoo capo: .mponuoz cw poaauomop mm mums mcoaumnaocH .nomo we 0H A we .mooHHm Hm>HH mo mmumoaaoou :c: you .z.fiwm a comma one moaam> am a mm m H cm s H mm m s cm N H m. m.H a H. m. H N.~ q.N H m.H m o Enhmm H Hooumv umoum a mo mooeam Ho>fia CH 09 mm pwo< muumm mo cowuouomm paw coaumofimauoumm co Aom no Bauom Sues Azmzv capo: Hmwucommm anagcfiz_m> Ammxv HowaHMnmnoux mo muoommm may .m manna 56 Table 6 demonstrates the effects of a combination of fatty acids, ie. palmitate, stearate, oleate and linoleate (equimolar quantities, .05 mM each) vs palmitate (.2mM) alone on esterification and secretion of fatty acids as TC in steer liver slices. Again, no TG were detected in the media in either case. There was a significant difference (p‘<.05) in esterification of fatty acid to TC in tissue due to the treatment combination of fatty acids. Table 6. Effect of Combination of Fatty Acids vs Palmitate Alone as Substrate for TG Esterification and Secretion in Steer Liver Slices. Media Tissue h n Palmitate Combination n Palmitate Combination ---------- pmoles/mg- - - - - - - - - - - - - - 4 4 1.7 i 2.7 .98 i 3 4 457 i 44 549 i 36 8 4 2.5 i 1.7 .52 i 1.5 4 512 i 181 745 i 88 Values are means i S.E.M. for "n" replicates of liver slices 60 i 21 mg each. Incubations were as described in Methods. Flasks were gassed every 90 minutes with 95:5 (0 :CO ) and final specific activities were .4258 pmoles/dpm (comb nation) and .34393 pmoles/dpm (palmitate). Zero times subtracted out. Media contained either .2mM palmitate or .05mM quantities of palmitate, stearate, oleate and linoleate. Labelled palmitate was used as tracer in both treatments. 57 Results in Table 7 show the effect of increasing the concentration of substrate fatty acid (palmitate) from .2mM to lmM in liver slices from a lamb. Consistent with previous results, no TG secretion was detected in the media. There was a significant (p< .001) increase in esterification of fatty acid to TG in tissue, although not directly proportional to the increase in substrate concentration. Esterification rates were doubled from 4 to 8 hours in both cases, indicating viable tissue function. Table 7. Effect of Fatty Acid Concentration .2mM vs lmM - on Esterification and Secretion of Fatty Acid as TC in Lamb Liver Slices. h n Media n Tissue -------- pmoles/mg - - - - - - - - - - - - - - - .2mM lmM .2mM lmM 4 4 -.43 i 1.8 -7.2 i 8.8 4 234 i 58 486 i 136 8 4 7.7 i 1.7 -4.17 i 1.5 4 542 i 41 1066 i 91 Values are means i S.E.M. for n replicates of liver slices, 80 i 22 mg each. Incubations were as described in Methods. Media contained either .2mM palmitate or lmM palmitate as substrate fatty acid. Flasks were gassed every 90 minutes with 95:5 (02:002) and final specific activities were .52169 pmoles/dpm (.2mM) and .47808 pmoles/dpm (lmM). Zero times subtracted out. 58 Another possibility for the reduced secretion rates in ruminant livers was suggested to be due to time between slaughter to incubation. All the studies with ruminant livers required anywhere from 25 to 35 minutes between slaughter to first flask incubation. The rats, on the other hand, are decapitated in the lab and livers are prepared for incubation within 10 to 15 minutes. Tables 8 and 9 demonstrate the effect of time from slaughter to first flask incubation on rat and steer livers. Results in Table 8 suggest that leaving the liver in the dead animal for 20 minutes, "slow" rat, was detrimental to the tissue as secretion was significantly lower (pc<.001) at 4 and 8 hours compared to the "fast" rat. It also appears, however, that esterification in tissue was affected as rates in the "slow" rat were significantly lower (p<:.001) than those in the "fast" rat and not significantly different between 4 and 8 hours. This latter observation is not seen in ruminants. Normally esterification rates are increased significantly over time in ruminant liver slices. This study suggests that the tissue in the "slow" rat was not viable. Following discussions with the manager of the slaughterhouse, an attempt was made to decrease time from slaughter to first flask incubation with a single steer. In fact, time was reduced to 9 minutes from slaughter to first flask incubation. However, results shown in Table 9 demonstrate that a reduction in time did not improve secretion of TG. Negative values indicate that zero time counts were higher than 4 or 8 hour period counts. Tissue 59 Table 8. Effect of Time from Slaughter to First Flask Incubation on Esterification and Secretion of Fatty Acid as TC in Rat Liver Slices. Media Tissue h n Fast Slow n Fast Slow ---------- pmoles/mg - - - - - - - - - - - - - - 4 4 5.6 i .7 1.2 i .6 4 416 i 76 119 i 35 4 27 i 2.7 2 i .8 4 877 i 130. 107 i 25 Values are means i S.E.M. for "n" replicates of liver slices, 54 i 15 mg each. Incubations were as described in Methods. Media contained .1mM palmitate and .1mM oleate and flasks were gassed every 90 minutes with 95:5 (02:C02). Final specific activity was .298 pmoles/dpm. "Fast" indicated that livers were removed immediately after decapitation (11 minutes to first flask incubation). "Slow" indicated a waiting period of 20 minutes before removing the liver for a total of 33 minutes to first flask incubation. Both rats were female litter mates and of comparable weights (555g). Zero times are subtracted out. Table 9. Effect of Decreased Time from Slaughter to First Flask Incubation on Esterification and Secretion of Fatty Acid as TC in Steer Liver Slices. h n Media n Tissue --------- pmoles/mg - - - - - - - - - - - - 4 4 -.32 i .9 4 168 i 15 8 4 -.75 i .7 4 344 t 28 I! H Values are means i S.E.M. for n replicates of liver slices, 93 i 27 mg each. Incubations were as described in Methods. Media contained .1mM oleate and .1mM palmitate and flasks were gassed every 90 minutes with 95:5 (02:00 ). Final specific activity was .1438 pmoles/dpm. Time from sIaughter to first flask incubation was 9 minutes. Zero times are subtracted out. 60 esterification doubled between 4 and 8 hours indicating viable tissue. All subsequent samples of ruminant liver slices were obtained rapidly and time from slaughter to first flask incubation was within 8 to 12 minutes. Also evaluated was the effect of pre-incubation holding temperature of liver slices on TC secretion. Normally all tissue is placed on ice for transport from the slaughterhouse to the lab. The possibility that cold holding temperatures may have affected the physical characteristics of the liver tissue enough to impede secretion was evaluated. Results in Table 10 demonstrate no effect of temperature on secretion or esterification of fatty acid as TC in steer liver slices. No TC was detected in the media, however, esterification rates were doubled from 4 to 8 hours and these rates were comparable to previous assays. Table 10. Effect of Preincubation Holding Temperature (2°C vs 25°C) in Esterification and Secretion of Fatty Acid as TC in Steer Liver Slices. h n Media n Tissue -------- pmoles/mg- - - - - - - - - - - - - - - 2°C 25°C 2°C ' 25°C 4 4 -4 i .7 .18 i 2 4 203 i 13 221 i 45 8 4 -.67i .6 l i .7 4 499 i 29 454 i 45 Values are means i S.E.M. for n replicates of liver' slices, 72 i 16 mg each. Incubations were as described in Methods. Media contained .1mM palmitate and .1mM oleate. Flasks were gassed every 90 minutes with 95:5 (02:C02). and final specific activity was .27405 pmoles/dpm. Zero times subtracted out. 61 Another important aspect of the development of this liver slice system was the need to verify that the TC secretion into the media was actually VLDL-TC and not an artifact of the system. Since TC secretion from ruminant livers was low and difficult to detect, a rat liver was repeated. In addition, the effect of insulin on TG secretion in the rat liver was also evaluated. Addition of puromycin (100ug/ml) to rat liver slices greatly reduced secretion (p<<.01) of TC at 8 hours compared to control and insulin treatments (Table 11 and Figure 1). This strongly suggests that the TG being secreted is accompanied by an apoprotein and is indeed VLDL-TC. Puromycin did not appear to affect esterification of TC in tissue (Table 11). Addition of insulin to rat liver slices tended to increase both TC secretion and esterification (Table 11). The results, however, were not statistically significant as the standard errors were quite high. The oleate to palmitate ratios were not different between treatments, however, in general it appeared that palmitate was secreted as TC in preference to oleate (Table 11) when both substrate fatty acids were present in equimolar quantities. 62 OF '0, .moo__m 32. you Eat oh no Boo >33 Lo cozocoom ozone: co c_o>Eoc:a uco £39: *0 aootm .P 0.52... A2205 2:: cosonaoc. m a a a . m n L P bN Inq— P ‘ £23.23". I .8300 Ola 5.3:. I 1! O m 7' I o o F fiw/selowd .oumuwaamo 28H. paw oumoao SSH. mmz taco huumm mumuumnnm .Emp\moHoEn Hence. mos oumoHOnmnm Mom was Emp\moHoEa mummm. mm3 oumuwaamnuUSHnH mo >ua>wuom oamaooam Hanan .ANOUNOV mama nuwz mouufiwa ca >Hd>o powwow ouo3 mxmmam .muonuoz aw Confluence mm mums meowumnnoaH .zomm we ma H mm .mooaam um>HH mo moumoaaoou :c: you wanna mo mocmuommwu mo Hound pumpcmum H mcmoa mum moaam> s s s s s m t mo.H ma. sa. as. as. am. m\o mam mama «so asaa sea“ mesa ama Hades mma HA5 ans mooa men one mom tumuaaata emu sow mss ssa Hsn Nam sms tumoso mDmmHH ma. mm.H as. an. as. as. m\o Hm sH ms mmfi on ssa ms Hmuou om a.a m.m OHH s.sH a.sm m.a tumuaaama am e a.a A.Ha s.mfl m.am s.m mumtao : n u u u u u u u u u u u u wE\mCHCEMn u u n u u u u u n n a u : em a . s w s m s Ass teat dwoafiousm . awanmsH Houucoo whet: .umm a mo mooHHm uo>HA Eoum 09 mm pao< muumm mo Gowumuoom can coquOHMHuoumm co Amy :Hohaousm use AHV aHHamaH mo Doommm .HH canoe 64 Two incubations were carried out with heifer (Table 12 and lamb (Table 13) liver slices in an attempt to induce lipoprotein secretion. The heifer liver slice experiment was designed to evaluate the effects of insulin (.1 U/ml) and/or estradiol (5 pg/ml) in the presence of palmitate alone or in combination with oleate (1:1 ratio). The final substrate fatty acid concentration was .2mM (.1mM palmitate + .1 mM oleate or .2 mM palmitate). Results in Table lzshow that total secretion of TC is again very low. However, estradiol (p'<0.1) and oleate (p‘<.01) increased TC in media. In addition, oleate increased esterification of TC in tissue. In the presence of both estradiol and oleate, the percent of TC in media vs tissue TC was greater (p‘<.05), perhaps a reflection of the increases in TC in media previously mentioned. The negative value seen with insulin treatment indicates that 6 hour values were less than zero time control values. There was also a significant positive correlation between media and tissue nmoles of TC and slice weight (.54 and .46, respectively) indicating a viable tissue. Finally, between .01 and .052 of the dose was found .in media TC and between 22 and 52 of the dose was found in tissue TC. The second assay utilizing liver slices obtained from a lamb was designed to evaluate the effect of triiodothyronine (T3) (100 ng/ml) and its interaction with insulin and estradiol on lipoprotein secretion. In addition, this assay utilized a dual labelling procedure 3H-oleate .uso nouomuundw mum moawu ouoN .Eap\mmaoem moosm. mm3 auH>Huom owmaommm Hmcwm .ANOUHNOV mumm nuHB mmuacHE om hum>o powwow oum3 mxmmam .muonuoz CH ponwuomou mm ouo3 mcoaumnnosH .nomo we Hm H we .mmoHHm Ho>HH mo moumoaamou :G: pom canoe mo moocouomwwp mo Hound pumpcmum H manna mum monam> 65 s m s s s s s m H t as. Hs.H HH.H ss. as. am. sa. sm. Ho.H H CH mamAHu Hoe «Huts smH son sHs Has mma mam saw HHH msu ma\thoaa mammaa as.H s.m Nm.~ ao.H ma. ss. ma. HH.H- sm.H maHthosa new m m H o m m H o o o o H H .Iallu «Hem: .aoauwnsocH mo mason me uo>o mmoHHm Ho>HA nomad: scum we we uHo< muumh mo coHuouoom new toHumuHHHutumm to eHo< Hos oHtHo eat Ame HoHemuutm .HHs :HHsttH Ho HomHHm .HH oHntH 66 muo3 mxmmam mum moanamm mEHu ouoN mm3 oumoaoumm How muH>Huom owwaooam Hanan .mponuoz CH ponwuommu mm ouoB maoaumnnocH mo mmumoaanmu :c: pom memos mo moocmuommap mo Hound pudendum H edema mum monam> .Bnp\mmaoaa mama. mm3 mum» .HNoou .uno pouomuunsm Sammnocaua you new Enu\mmHoEQ mnmmo. ov mama :uH3_mouacHE om >uo>o powwow .nomo we NH H as mooHHm Ho>HH s s s s H m s s t H.Hs H.HHH H.sHH HHH sOH HHH m.mHH s.msH H.HNH HHmHous mSHCtHoaa s.- m.ms H.as am an m.nm H.ss s.ma H.OH AcusHs HSHAtHoaa m.wH w.Hs a.sm m.as H.ss H.Hs s.Hm m.sa mm AH mHs wSHthoaa mammHH HH.s mm.m am.s Hm.s ss.H s~.m HH.H mo.~ .HH.H HHtuous wEHCtHasA ~H.H HH.H ms.H ms.H aa.H HH.H mH.H mm. m~.H Ao.sHs wSHthoaa HH.H Ho.s Ha.~ mm.H ms.n Hm.H sm.~ -.H as. 1H.mHs wEHthoaa Hm H m H H H m H o .<.H m me my H me .111: «Hem: .aowumnsocH mo «Mao: me Ho>o mooaam Ho>HA gang Scum we no pHo< muumm mo coaumuoow use coaumowwauoumm do Amav ocficonmSuOCOHHHH was Amv smwouumm .AHV :HHDmaH mo uommmm .mH oHan 67 and 14 C-palmitate along with .1mM total palmitate and .1mM total oleate as substrate fatty acids. Results shown in Table 13 again demonstrate that TC secretion into the media is low, consistent with previous ruminant liver slice assays. Addition of T3 to the media tended to increase TC in media and decrease esterification of TC in tissue. Insulin increased TC from oleate and palmitate in tissue (p<=0.l). In this assay there was a weak correlation of tissue weight to media TC (-.19 to + .13) suggesting no TC secretion into the media. Correlation between tissue weight and tissue esterification was similar to the previous assay (.43) suggesting the tissue was viable. Percent of dose in media (.02 to .072) and tissue TC (1.2 to 2.12) was comparable to the previous assay. Species Comparison In ViéW’Of the consistently low TC secretion observed in ruminant liver slices, the idea that the potential for "de novo" hepatic fatty acid synthesis might be a prerequisite for secretion of TC developed. In an attempt to evaluate this hypothesis, a number of species were selected based on their relative capacity for "de novo" hepatic fatty acid synthesis. Studies have shown the liver to be the predominant site of lipogenesis in avian (Yeh and Leveille, 1969, 1970), fish, (Lin et al., 1977c) and humans (Shrago et al., 1971), while adipose is the predominant site of lipogenesis in ruminants (Hanson and Ballard, 1967), pigs (O'Hea and Leveille, 1969b), guinea pigs (Patel and Hanson, 1974), and dogs (Baldner et al.,1985). 68 Both adipose and liver tissue are responsible for a major proportion of the lipogenesis that occurs in rats (Leveille, 1966), mice (Muiruri and Leveille, 1970), and rabbits (Leung and Bauman, 1976). Of the above species, liver slices were incubated from chickens, fish, rats, rabbits, pigs, guinea pigs, sheep and cattle in order to compare TC secretion. Media contained .1mM oleate and .1mM palmitate as fatty acid substrate. Initial comparative studies (Table 14) between the rat and pig (using dual labelled techniques) appear to be consistent with the previously mentioned hypothesis. Secretion of TC into the media from pig liver slices was not detected until after 8 hours. The 12 hour mean is also questionable as individual values for each flask varied widely (.82, 2.53, 12.5 and 61.2 pmoles/mg). In the rat, however, secretion rates were linear up to 12 hours. Regarding esterification of fatty acid to TC, results were similar between the two species. Esterification rates were linear up to 12 hours and if total esterification (ie TC secreted + TC in tissue) is compared, there are no differences between the rat and pig. A second pig trial (Table 15) demonstrated similar results. TC secretion into the media was not significantly different from zero at 4 or 8 hour incubation periods. Esterification, however, was doubled between 4 and 8 hours and rates were similar to the previous pig liver slice assay. Only one assay with a male albino guinea pig was carried out (Table 16). Secretion of TC into the media was 69 mew wwEHu oewu .ueo nwuoweunew .uwe Hewewweeeoe onEwm w cum w new ewon wx maa w wewB nwwe wHwEHee ..ouwuHEHwasoanH mo Een\wwHoEe mmmmm. new wuwwHOucmmo Een\wwHoEm Hmooo. wws >uH>Huow cameowaw Hwewm new ANOUHNCV mama £uw3 wwueewa om zew>w nwwwww mews mxwwam nwnHeowwn mw wew3 weoHuwneoeH .meoebtz tH .Humus auto we on H sm eat AwHas comm we HH H as .mtoHHm Hw>HH mo wwuwowaawu :e: Mom wewwE mo woewewmwwn mo uouew newnewuw H wewwE mew wweaw> s s m s m s t 0mm Hos mam Ham AHHH wHH sss Httou HHH HHH HHm aHH cam sms mHH tuttHeHma HHH Ham HHH NsH HHs mmH HHH muttHo wewwHH Hm HmH H.ma H.Hm H.HH H.o- s.m- Htuou w.mH m.oa o.Hs H.sH H.AH a.H- o.H- mHmHHsHma s.mH H.os H.mH s.H w.H a.s- s.s- tutho «Hem: n a u u u u u u n n u u u u u u u u : wE\meoEm : u n u u u a u n u n mm a NH 5 w e s a HH a m a s Hes teHu wmm mam mam w> uwm ea UH ww eowuweowm new eoHuonmaeouwm nHo< Muuwm mo eowHuweEoo 4 .sH «Heme 70 Table 15, Esterification and Secretion of Fatty Acid as TC in Liver Slices of a Pig. h n Media ' n Tissue --------- pmoles/mg - - - - - - - - - - - - - - 4 4 .17 i .7 4 395 i 68 8 4 3.7 i 1.3 4 945 i 63 Values are means i S.E.M. for n replicates of liver slices, 56 i 8 mg each. Incubations were as described in Methods. Media contained .1mM oleate and .1mM palmitate as substrate fatty acid. Flasks were gassed every 90 minutes with 95:5 (02:C02) and final specific activity was .35830 pmoles/dpm. A 127 kg boar was used as source of liver. Zero times are subtracted out. Table 16, Esterification and Secretion of Fatty Acid as TC in Liver Slices of a Guinea Pig. h n Media n Tissue ---------- pmoles/mg - - - - - - - - - - - - - 4 4 -l.2 i .8 4 406 i 78 8 l 4.6 4 834 i 38 H '0 Values are means i S.E.M. of n replicates of liver slices, 55 i 11 mg each. Incubations were as described in Methods. Media contained .1mM oleate and .1mM palmitate as substrate fatty acid. Flasks were gassed every 90 minutes with 95:5 (02:CO ) and final specific activity was .34632 pmoles/dpm. A 427 g male albino guinea pig was used as the source of liver. Zero times are subtracted out. 71 also negligible at 4 hours and 8 hours (only one 8 hour replicate due to laboratory error) again consistent with the hypothesis. Esterification was doubled between 4 and 8 hours and was similar to the previous results in pigs. Triacylglycerol secretion studies from ruminant liver slices exposed to similar media conditions (.1mM oleic and .1mM palmitate) have been referred to (Tables 9 and 10). In addition, slight variations in media substrate fatty acid (.2mM palmitate as sole source) gave similar results (Tables 3-7, 12 and 13). Lipogenesis in rabbits occurs in both adipose and liver (Leung and Bauman, 1976). Combined results of liver slice assays from one female and two male rabbits (Table 17) clearly demonstrate secretion of fatty acid as TC. Also noted was a difference in secretion between the female rabbit and the two male rabbits at the 8 hour incubation period (60 i 8, female; 26 i 10, male - data not shown in Table 17). Esterification rates doubled from 4 to 8 hours and were similar to results of previous assays. The lipogenic pattern in rats is similar to that in rabbits and secretion of fatty acid as TC has been reported in Tables 2, 8, 11 and 14. The last two species to be considered are avian and fish (Table 18 and 19). The combined results of liver slice assays from three White Leghorn laying hens demonstrate that TC secretion is increased over that of rats and rabbits (Table 18). Finally, results of the study utilizing trout liver 72 Table 17. Esterification and Secretion of Fatty Acid as TC in Liver Slices of Rabbits. h n Media n Tissue -------- pmoles/mg - - - - - - - - - - - - - - 4 3 4 i 1.3 3 542 i 15 8 3 39 i 12 3 1156 i 24 Values are means i S.E.M. for n replicates of liver slices, 56 i 13 mg each. Incubations were as described in Methods. Media contained .1mM oleate and .1mM palmitate as substrate fatty acid. 'Flasks were gassed every 90 minutes with 95:5 (02:C02) and final specific activity was .35417 pmoles/dpm. Data are combined results of 2 male and 1 female New Zealand White rabbits. Zero times subtracted out. Table 18. Esterification and Secretion of Fatty Acid as TC in Liver Slices of Single Comb White Leghorn Laying Hens. h n, Mediaii n, Tissue --------- pmoles/mg - - - - - - - - - - - - - 4 3 49 t 20 3 496 i 109 3 180 i 84 3 1073 i 205 Values are means i S.E.M. for n replicates of liver slices, 62 i 19 mg each. Incubations were as described in Methods. Media contained .1mM oleate and .1mM palmitate as substrate fatty acid. Flasks were gassed every 90 minutes with 95:5 (02:002) and final specific activity was .310559 pmoles/dpm. Results are combined data of 3 hens. Zero times subtracted out. 73 Table 19. Esterification and Secretion of Fatty Acid as TC in Liver Slices of Trout. h n Media n Tissue -------- pmoles/mg - - - - - - - - - - - - 4 3 43 i 16.4 3 334 i 69 8 3 107 i 25 3 437 i 73 Values are means i S.E.M. for n replicates of liver slices, 58 i 10 mg each. Incubations were as described in Methods. Media contained .1mM oleate and .1mM palmitate as substrate fatty acid. Flasks were gassed every 90 minutes with 95:5 (02:C02) and final specific activity was .35014 pmoles/dpm. Results are the combined date of l Brook and 2 Rainbow Trout, all females and of comparable size. Zero times subtracted out. 74 ' Slices (Table 19) are similar to studies with avian liver slices. Secretion of TC at 8 hours was doubled compared to the 4 hour period. In this study, the incubation temperature was maintained at 20°C (Rogie and Skinner, 1985) as these are poikilothermic species viable in water at temperatures ranging from 0°C to 20°C. Results support the hypothesis that the capacity for hepatic lipogenesis is related to the ability to secrete TC. Two final attempts were made to see whether the capacity for ruminant hepatic TC secretion might be related to lipogenic capacity by evaluating ruminants at different levels of maturity. Secretion of TC from liver slices of a bull calf (Table 20) was very low and hardly detectable. Esterification of fatty acid to TC was more than doubled from 4 hours to 8 hours and the tissue appears to have been viable. Due to the difficulty in obtaining a fetal ruminant, a neonatal lamb, 2 hours old and not given to suck was secured. In all replicate flasks but one at 4 hours, positive secretion values were obtained (Table 21). However, secretion was still very low and comparable to earlier ewe studies (Table 3). Tissue esterification doubled over the 4 hour to 8 hour period and the tissue appears to have been viable. A summary of the results on the species comparison studies is shown in Figure 2. The bar graph represents the combined data of the individual species. Not depicted in the graph is the variation within groups and within species. As discussed previously, there is some variation due to sex, stage of production and diet. This study cannot begin to 75 Table 20, Esterification and Secretion of Fatty Acid as TC in Liver Slices of a Functional Nonruminant. h n Media n Tissue --------- pmoles/mg - - - - - - - - - - - - - 4 4 -.31 i .6 4 169 i 21 8 4 3.4 i 2.7 4 400 i 31 Values are means i S.E.M. for n replicates of liver slices, 87 i 16 mg each. Incubations were as described in Methods. Media contained .1mM oleate and .1mM palmitate as substrate fatty acid. Flasks were gassed every 90 minutes with 95: 5 (02: C02) and final specific activity was .35638 pmoles/dpm. The liver slices were obtained from a two week old Holstein bull calf. Zero times are subtracted out. Table 21. Esterification and Secretion of Fatty Acid as TC in Liver Slices of a Neonatal Lamb. h n Media n Tissue ------- pmoles/mg - - - - - - - - - - - - - 4 4 3.5 i 2 4 294 i 16 8 4 3.5 i 1.3 4 510 i 15 I! I! Values are means i S.E.M. for n replicates of liver slices, 60 i 15 mg each. Incubations were as described in Methods. Media contained .1mM oleate and .1mM palmitate as substrate fatty acid. Flasks were gassed every 90 minutes with 95: 5 (02: C02 ) and final specific activity was .38752 pmoles/dpm. Samples were obtained from a crossbred lamb neonate within 2 hours of birth. Zero times are subtracted out. 76 A W V C 351 g E .C L.) 30- 25‘ <3) 7 f2 "3. 20- “L H. I? _- § 15~ g (3) (4) D- H 10- 2 C .D .D (2) (1) <5) (9) g g 5- 2 a 2 cs '3‘ 3 3 W . .C U m u, w u OJ $ 353 $1 & I o 50 ‘160 Percent 2 Figure 2. Relative capacity for hepatic lipogenesis versus hepatic secretion of fatty acid as TG. Numbers in parenthesis represent number of experiments. Histograms are means i S.E.M. for (n) replicates. Incubations were as described in Methods. Media contained .2mM fatty acid. Flasks were gassed every 90 minutes with 95:5 (02:C02). 77 answer these specific differences nor was it designed to. However, the graph does depict an observable species difference in the ability to secrete fatty acid as TC in this in vitrg liver slice system. This difference appears to be related to the lipogenic capacity of the liver. DISCUSSION EN VITRO ASSAYS Measurements of TG secretion as VLDL in 21339 have been carried out predominantly in rats and avian species. These studies have utilized liver explants, hepatocytes and perfusion systems, along with a variety of media for the incubations. No studies of this sort have been carried out in ruminant species, therefore, no information on appropriate systems or media is available. Minimal Essential Media (MEM) with Earle's salts and 25 mM Hepes buffer and without L- glutamine was selected as the media of choice based on studies in rats (Jauhiainen, 1983). Additionally the ease of obtaining the commercially prepared media, sterilized and ready for immediate use and its recommended storage life of up to 4 months at 2°C to 8°C (doubled by withholding L- glutamine) made it very accommodating. Earle's salts were selected over Hank's, as they are best employed in culture systems which are tightly closed or, if open, exposed to an atmosphere containing about 52 002 (Paul, 1972). A final fatty acid concentration in the media of .2mM was selected, based on previous studies with perfused livers of rats (Laker and Mayes, 1981; Heimberg et al., 1978 and Olubadewo et al, 1983), bovine liver homogenates (Benson and Emery, 1971), and ovine liver slices (Payne and Masters, 78 79 1971). Palmitate was chosen as the substrate fatty acid due to its more stable nature compared to unsaturated fatty acids and it represents up to 25% of blood NEFA in the ruminant (Bickerstaffe et al., 1974). Liver slices have been used in the past to measure rates of fatty acid esterification in rats (Rose et al., 1964; Rubenstein and Rubenstein, 1966) and sheep (Payne and Masters, 1971). Tissue esterification rates obtained in this study (Tables 2 and 3) were comparable to the above mentioned studies amounting to about 22 to 52 of the labelled fatty acid in the media incorporated into tissue TG. Rates of TG secretion from liver slices have not been reported. The average rate of TG secretion in rats in this study (Figure 2) (7.5 pmoles.mg-1. h'l) is lower than (60 to 200 times) rates reported for perfused livers (Goh and Heimberg, 1977; Azain et al., 1985). Results of TG secretion in isolated hepatocytes of rats appear to be less than 252 of perfused livers (Laker and Mayes, 1984). While TG secretion from liver slices in the present study are low compared to other systems, the relative differences between species using liver slices are marked (Figure 2). The species differences was also reflected in the contamination problem that occurred in the laboratory analysis. During the initial analytical procedures labelled fatty acid appeared to contaminate the TG spot on the TLC plates. This affected the zero time samples and resulted in fairly high blanks. In order to minimize this contamination, the extraction procedure was made more basic to avoid 80 partition of excess labelled fatty acid. In addition, the total dpm per flask was reduced by 252 to 1 x 106 dpm/flask and the amount of activated alumina (a fatty acid chelator) in the extraction was increased from .5'g to 1.6 g reducing the fatty acid contamination. The negative values for T6 secretion that appear in several of the tables are a reflection of the low secretion that occurred consistently in the liver slices of the respective species. Tables 3 through 15 describe the deveIOpment of the liver slice system in the ruminants. Initially, no attempts were made to develop the liver slice system using type of ruminant, age, sex, etc. In general all attempts to measure TG secretion resulted in low secretion. For that reason, livers from ruminants as a class were evaluated. In view of the very low secretion of TG by the ovine liver slices, (Table 3) subsequent assays attempted to evaluate whether there may have been something missing in the media which was limiting TG secretion in ruminant livers. Glucose is not an important substrate for lipogenesis in the ruminant liver and in fact is produced and exported by the liver for use in extrahepatic tissues (Ballard et al., 1969). Approximately half the glucose in ruminants is made from propionic acid, an end product of rumen fermentation (Leng et al., 1967). In view of the importance of prOpionic acid for hepatic gluconeo- genesis (GNG) it was added to the media as a possible limiting factor to normal hepatic metabolism. Addition of propionate to the media did not appear to enhance secretion 81 of TG (Table 4) in steer livers. The observed esterification rates, .1 nmole h.”1 mg."1 were lower than those of 15 nmoles h.-1 mg.-1 in bovine liver homogenates (Benson and Emery, 1971). These differences may be due to increased exposure of enzyme sites to fatty acid following homogenization of tissue resulting in more rapid rates of esterification. Addition of serum in the presence of either Minimal Essential Media (MEM) or a Krebs Ringer Bicarbonate buffer (KRB) also appeared ineffective in enhancing TG secretion. It was suggested that formation of tripalmitin (melting point, 66°C) might preclude secretion in a system at 37°C if the TG solidifies following esterification. Additionally, oleate has been shown to increase secretion of TC in perfused . rat liver compared to palmitate (Goh and Heimberg, 1977). Adding a combination of fatty acids at equimolar concentrations (.05 mM each of palmitate, stearate, oleate and linoleate) did not enhance TG secretion in steer liver slices (Table 6). The significant increase in fatty acid esterification in tissue is not readily explained as others have shown saturated fatty acids, especially palmitate to be esterified in preference to unsaturated fatty acids in liver slices (Heimberg, et al., 1974). The complex series of reactions leading to secretion of TC is a saturable process and increasing fatty acid concentration can increase TG output up to a point. Augmenting the infusion rate of fatty acid from O'to 331, 663 and 1326 umoles of oleic acid over 4 hours in a perfused 82 liver resulted in linear output of TG reaching saturation at the highest level of infusion (Goh and Heimberg, 1977). However, attempts to increase secretion of TG from liver slices of the ruminant by increasing fatty acid concentration from..2mM to lmM did not enhance TG secretion into media (Table 7) although tissue esterification was increased. In some cases the physical handling of the tissue prior to incubation may influence results. As indicated in Tables 9 and 10, decreasing the time from.slaughter to first flask incubation or maintaining the tissue at room temperature vs. cooling it on ice prior to slicing and incubation, did not affect rates of TG secretion. The results in Table 8 with the rat, however, imply that the time from slaughter to incubation is important. The ?slow" rat liver was left in the rat for 30 minutes prior to slicing and incubation. This appeared to be long enough to affect tissue viability as esterification rates were reduced and not different between the 4 and 8 hour periods. It would seem that the ruminant livers were not affected by this time element as the tissue esterification rates were not similarly impaired in any of the studies. I The use of puromycin is an indirect method to determine that the TG that is being secreted is in fact accompanied by apoprotein and is likely to be VLDL. It has been demonstrated that secretion of VLDL is totally dependent on apoprotein B (Kane, 1983). Puromycin and cycloheximide are both protein synthesis inhibitors and have been shown to inhibit VLDL 83 secretion by preventing formation of apoprotein B (Janero and Lane, 1983; Davis et al., 1982; Kempen, 1980). Siuta- Mangano et al., (1982) have demonstrated that ongoing apoprotein synthesis is also necessary for hepatic VLDL secretion, therefore factors affecting protein synthesis are more critical. The observation that tissue esterification rates did not appear to be affected may be due to a postulated cytosolic resevoir of enzymes (Butterwith et al., 1984). It has been suggested that a resevoir of enzymes involved in the esterification of fatty acid may be harbored within the cytosol, available to respond to fatty acid mobilized from adipose in times of stress (Pittner et al., 1985). The high rate of TG synthesis protects the liver against the toxic effects of fatty acids. This resevoir of enzymes might explain the lack of effect of puromycin on tissue esterification (Table 11) in this short term incubation. The possibility that secretion of TG would be enhanced by adding hormones to the media was also evaluated in the ruminant liver slices. Responses to insulin addition have been variable and not well understood. Studies in perfused rat livers have shown increases in VLDL secretion with insulin addition (TOpping and Mayes, 1972; Laker and Mayes, 1982); and Heimberg et al., 1974). In contrast, isolated hepatocyte preparations have demonstrated either no effect (Mangiapane and Brindley, 1986; Beynen et al., 1981) or a decrease (Pullinger and Gibbons, 1985; Patsch et al., 1983; and Durrington et al., 84 1982) in VLDL secretion. The lack of a positive response to insulin in isolated hepatocytes from rats has been attributed to inherent problems within the hepatocyte system, which released TG at less than 252 of that reported for perfused livers (Laker and Mayes, 1984). Moreover, the system.was suggested to be unsuitable for studying dynamic events in the liver that depend on an active blood supply for maintaining physiological rates of metabolism (Laker and Mayes, 1984). Chick hepatocyte cultures, on the other hand, require insulin for VLDL secretion and removal of insulin causes a parallel decline in acetyl CoA carboxylase activity and VLDL synthesis (Tarlow et al., 1977). The increase in TG secretion with insulin in perfused livers has been attributed to short term control of hepatic lipogenesis by covalent modification of acetyl CoA carboxylase (Beynen et al., 1979; Lee and Kim, 1977) or alternatively long term control via enzyme induction of lipogenic enzymes (Lakshmanan et al., 1972). The end results of both of these controls would be to increase substrate TG for VLDL formation. Any lipogenic effects of insulin would probably not be of great consequence in the ruminant liver as "de novo" lipogenesis is of minor importance (Ballard et al., 1969). Therefore, if insulin affected secretion independent of increases in lipogenesis, it might have been detected in this system, however, insulin did not induce secretion of VLDL in ruminant liver slices (Tables 12 and 13). There did appear to be a slight though nonsignificant response to insulin in rat liver 85 slices (Table 11) which is consistent with the stimulation of lipogenesis as previously discussed. In regards to the effects of estradiol and T3, the length of incubation is more critical. Estradiol and T3 normally affect nuclear receptors at the level of the genome resulting in increases in protein synthesis (Levey and Robinson, 1982). The increase in protein synthesis may not ShOW' up until after 6 hours (Beynen et al., 1979). This would require incubation periods much longer than the 6 hours in these assays (Table 12 and 13) to see an effect. Previous studies with estradiol have either pretreated donor animals (Miller and Lane, 1984; Weinstein et al., 1986) or pre-incubated tissue up to 12 hours prior to initiation of the experiment (Goodman et al., 1983). Similar methods have been used for T3 (Olubadewo et al., 1983; Olubadewo and Heimberg, 1985; Calandra and Tarugi, 1984; and Goodman et al., 1983). It might have been possible to see a response to these hormones had the animals been pretreated before removing the livers. Alternatively, a cultured hepatocyte system, which remains viable for longer periods of time, would be a better system to use for studying long-term responses. Hepatocytes could be pre-incubated with the hormones for 12 to 24 hours before attempting to measure a response. The increased TG secretion seen in Table 12 with oleate is consistent with studies in perfused rat livers (Goh and Heimberg, 1973 and 1977), and in isolated rat hepatocytes 86 (Davis and Boogaerts, 1982) where oleate addition stimulated VLDL secretion. This affect did not appear to be directly related to increases in tissue TG esterification (Davis and Boogaerts, 1982). Additionally, the increase in secretion was specific for TG and PL constituents only and did not affect apoprotein, cholesterol or cholesterol ester secretion (Davis and Boogaerts, 1982). Again, the most consistent observation in these studies to this point was the relatively low rate of TG secretion in ruminant liver compared to that observed in the rat. In view of the results just mentioned, a number of species comparison studies were carried out to see whether the low secretion rates may be species dependent. As mentioned previously, studies have shown that the predoninant tissue site of lipogenesis varies between species and the possibility that secretion of TC is related to this observation is suggested by results in our studies (Figure 2). Because the relative differences in secretion of TG were quite large between species in which liver lipogenesis is predominant compared to those in which it is fairly limited, no attempt was made to classify liver slices by gender. A sex difference in TG secretion has been reported previously in rats (Heimberg et al., 1974; Haagsman and Van Golde, 1984) with females secreting more VLDL-TG than males. This difference has been attributed to effects of estrogen as this hormone has been shown to induce TG secretion (Weinstein et al., 1986; Miller and Lane, 1984; Soler-Argilaga and Heimberg, 1976). 87 In regards to results with avian species, there appeared to be a difference in secretion due to production level. Because liver slices from only three hens were used and only approximate production records were available, only general observations could be made. Hen #1 was at about 60% production (365 d x .6 = 219 eggs/yr), whereas hens #2 and #3 were out of production. The liver of hen #2 was very fatty and yellow in color and contained about 26% fat (wet basis), whereas hen #3 had a firm red, normal looking liver containing 102 fat (wet basis). Individual TG secretion rates for the three hens at 8 hours were: #1, 340 i 59 pmoles/mg; #2, 150 i 20 pmoles/mg; and #3, 50 i 2 pmoles/mg. Mature hens do secrete more TG than immature hens (Bacon et al., 1978) and the increase has been attributed to estrogen (Kudzma et al., 1975) which is increased at the onset of production. Upon reaching sexual maturity, the estrogen influence may cause liver fat to accumulate up to 40% to 702 on a dry weight basis (Ivy and Nesheim, 1973). This increase in lipogenesis may lead to fatty liver hemorraghic syndrome, a chronic problem in the layer industry (Haghighi-Rad and Polin, 1981). The fatty liver which develops in the ruminant is normally in response to a negative energy balance, resulting in increased uptake and esterification of preformed fatty acids. In contrast, the avian fatty liver occurs in a positive energy balance and is due to increased lipogenesis within the liver (Couch, 1956). This may be a reflection of the lipogenic capacity of the liver in the two species. 88 The species difference is also reflected in literature values for activity of various enzymes involved in hepatic lipogenesis. Compared to the rat,(Baldwin et al., 1966) activities of glucose 6-phosphate (G6-P) and 6-phosphogluconate (6-G-P)were 10 to 50 fold lower in the guinea piglet, piglet and calf, while activities of citrate cleavage enzyme (CCE) were 20 fold lower in the piglet and calf. Malic-enzyme (ME) was much lower in the calf (below assay sensitivity) compared to the rat. Activities of CCE and ME were not reported for the guinea piglet and ME was not reported for the piglet. Similarly reduced activities of CCE, G-6-P and 6-G-P, have been reported in swine (O'Hea and Leveille, 1969b) and ruminants (Ballard et al., 1969). The hepatic lipogenic enzyme activities in rats, chickens and fish are more comparable. With minimal fat in the diet, activities of ME and CCE in chickens were from 48 to 64 and 42 to 55 (nmoles of substrate converted to product min.'1 mg protein-1) respectively (Yeh et al., 1970). This compares to a range of 65 to 87 and 16 to 30 (nmoles substrate converted to product min."1 mg protein-1.) for ME and CCE, respectively in coho salmon (Lin et al., 1977). Romsos et al., 1974, has reported ME activity in rats to be about 29 nmole substrate converted to product min.“1 mg protein-1. The activity of the NADPH generating HMP enzymes in salmon (Lin et al., 1977) are on the order of 10 to 100 fold greater than those observed in pigs (O'Hea and Leveille, 1969b) and ruminants (Baldwin et al., 1966). 89 Previous studies have shown that the fetal ruminant liver does have the capacity to synthesize lipid from glucose available from the maternal circulation (Hanson and Ballard, 1968). Levels of lipogenic enzymes are increased in the fetal ruminant several fold compared to the adult, although they are still much lower than levels in the fetal or adult rat (Ballard et al., 1969). The capacity to utilize glucose carbon for lipogenesis appears to end shortly after birth with the acquisition of rumen microflora (Ballard et al., 1969). Based on these studies, liver slices were obtained from a two-week old, milk fed bull calf, presumably a functional non-ruminant. However, no significant response in TG secretion was observed. It may be that although it was a functional nonruminant, the lipogenic capacity was still too low. Alternatively, in rats, it has been demonstrated that the high rate of lipogenesis in the fetal rat is decreased rapidly at birth, commensurate with the high fat milk diet. This is followed by a sharp increase at weaning when the rat begins to eat a carbohydrate diet (Ballard et al., 1969). It could be that the high fat milk diet in the bull calf depressed any lipogenic enzyme activity that might have been present in the fetal state. In an attempt to further evaluate this relatipnship between utilization of maternal glucose and lipogenic capacity by the liver, a neonatal lamb was obtained. The low rates of secretion observed in the neonate may have been due to a decrease in lipogenic enzyme activity compared to the fetal ruminant or 90 the level of enzyme activity may have been too low to detect increases in TO secretion. Also, as mentioned earlier, the level of lipogenic enzyme activity in the fetal ruminant is still very low relative to the fetal or adult rat (Ballard et al., 1969). Others have attempted to stimulate ruminant hepatic lipogenesis by chronic iv. glucose infusions. Measured activities of hepatic lipogenic enzymes fall far short of rates observed in rats (Ballard et al., 1972). Other researchers have attempted to correlate hepatic lipogenesis to hepatic secretion of TG in livers of rats (Windemueller and Spaeth, 1967). These investigators used livers from rats that had been fed ad libitum or were fasted and refed a high carbohydrate diet. Results of liver perfusion studies demonstrated a correlation of .83 between hepatic synthesis of fatty acid "de novo" and rate of release of TG. Others have also shown similar results with livers from rats under perfusion (Topping and Mayes, 1972) or I hepatocyte systems (Beynen et al., 1981). Recent studies by Pullinger and Gibbons, (1985) demonstrated this relationship to be true under certain conditions in rat hepatocytes, but also demonstrated that this relationship is by no means obligatory to TG secretion. Harvesting hepatocytes from rats that had been fed for 6 hours (D-6) resulted in higher rates of lipogenesis, but not significantly higher rates of secretion compared to hepatocytes from rats harvested before feeding (D-O). In another case, the opposite lipogenic response to pyruvate and glucagon in the D-0 and D-6 91 hepatocytes was not accompanied by significant corresponding differences in VLDL-TC secretion. Lastly, insulin, shown to stimulate lipogenesis, actually resulted in a decrease in VLDL-TC secretion. Azain et al., (1985) attempted to determine the contribution of "de novo" hepatic fatty acid synthesis to VLDL secretion in perfused livers from lean and obese rats. Following a calculation taking into account the intra hepatic turnover of the newly synthesized fatty acids, the contribution of "de novo" fatty acid synthesis to TG secretion was determined to be 9% in the lean and 44% in the obese rat liver. Previous studies by the same group (Fukuda et al., 1982) suggested that preformed fatty acid contribution (plasma NEFA uptake) accounted for 90% in lean and 56% in obese rat livers after accounting for turnover within the liver pool of TG. These results suggest that preformed fatty acids contribute more than "de novo" synthesized fatty acids to VLDL in normal rats. However, in obese rats, the two sources of substrate are of almost equal importance. The above mentioned study (Azain et al., 1985) utilized a perfusion media containing 25 mM glucose and .15- .20 mM oleic acid. These concentrations were designed to mimic in yigg conditions. This level of plasma NEFA has been shown to inhibit hepatic "de novo" fatty acid synthesis (Heimberg et al., 1978; Topping and Mayes, 1982). Therefore, the degree to which lipogenesis is inhibited by plasma NEFA will have a significant effect on the percentage contribution of these two VLDL substrates. In addition, actual contribution 92 in vigg is likely to vary considerably depending on diet and physiological state. In summary, the results of the present studies demonstrate a consistent species difference in the ability to secrete hepatic TC in this liver slice system. Moreover, this difference appears to be related to the relative lipogenic capacity of the liver. The possibility that the media lacks some factor required for the secretory process does exist, but in view of the consistent species response, it becomes less tenable. FATTY ACID KINETICS IN LACTATING COWS 93 MATERIALS AND METHODS Lactating Cows This study, partially supported by the Monsanto Company (St. Louis, Missouri) was designed to evaluate the hypothesized lipotrophic effects of methionine on endogenous TG secretion. Seven cows, 5 from the O.A.R.D.C. isotope herd and 2 acquired from the Ohio State University (O.S.U.) herd were utilized for the study. Cows were allotted to control or experimental diets during the dry period. The experimental cows were fed supplemental methionine hydroxy analogue (MHA 30 g/day) from approximately 2 weeks prepartum through the entire study. Diets of both groups were identical in every other respect (see Table 22, consisting of 50% concentrate, 25% corn silage and 25% haylage (DM basis), fed ad libitum during lactation (2 x daily). Feed intakes and refusals were recorded on the barn computer. At approximately 30 and 60 days of lactation, cows were continuously infused with palmitic acid l-lAC (approximately 6uCi/min) for 160 minutes to achieve steady state labelling of the body NEFA pool (precursor to endogenous VLDL-TC). Each cow was to be infused twice (at 30 and 60 days of lactation). This was not accomplished in every case, due to various problems with the cows. Two days prior 94. 95 Table 22. Composition of concentrate fed to cows. Ingredient % Ear corn, ground 47 Oats, ground 20 Molasses, dried beet pulp 10 Soybean meal 10 Corn gluten meal 8 Unifat M937 2 Dicalcium phosphate 1 Limestone .8 Trace mineral salt .5 Magnesium oxide .2 Selenium premix .1 Vitamin A .1 Vitamin D .1 Vitamin E .2 MHA3 .25 aMBA, methionine hydroxy analog - added to treatment diet (Monsanto Co.). 96 to infusion of tracer, the cows were weighed and moved to the radiotracer laboratory (air conditioned, 21°C) to become acclimated to the surroundings. The day before the infusion, 16 gauge polyvinyl- cholride (PVC) cannulae were inserted into both external jugular veins under local anaesthesia (Lidocaine). Experimental Procedure The day of the infusion, following the morning milking, a urinal was attached to collect radioactive urine. The isotope reservoir was attached to the infusion pump (Technicon Auto Analyzer Proportioning Pumps, Tarrytown, New York) which in turn was attached to the infusion cannula. Immediately prior to the infusion, the cow was milked, with the aid of oxytocin and a preinfusion blood sample was obtained. The infusion dose (1mCi of 1-14-C-palmitate 12.9 mCi/mmol; obtained from.New England Nuclear, Boston, Mass.) was prepared by adjusting the palmitate to a pH of 9 with NaOH and dissolving in 300 ml of distilled water overnight at 50°C. The purity of the labelled palmitate was checked by TLC and scintillation counting and determined to be greater than 99% pure. The concentration of activity was determined before and after infusion, and the amount infused was accurately determined by weighing the dose flask before and after infusion. All experiments were carried out at the same time of day and all the cows were on the same daily feeding ' schedule. Blood samples (10 and 40 ml) were collected at 20 97 minute intervals during the infusion. After the infusion was stopped, 40 ml collections were made at 5, 10, 15, 20, 30, 40, 60 and 90 minutes and again at 18 hours. Forty ml tubes contained ethylenediaminetetracetic acid (EDTA; 1 mg/ml) and protamine sulfate, a lipoprotein lipase inhibitor (.5 mg/ml). Ten m1 samples containing sodium flouride (1 mg/ml) were used to determine blood C02 specific activity. All blood samples were placed on ice until plasma was separated by centrifugation at 18,000 x g at 4°C for 20 minutes. Total lipid extractions were carried out the same day of the infusion. Milk samples were collected with oxytocin just prior to infusion and at approximately 3 and 9 hours post-infusion. Milk sampling, 2 x daily without oxytocin, was continued for 10 - 14 days post-infusion. Laboratory Analysis. Total lipids were extracted from 10 ml plasma according to the method of Cham and Knowles, (1976) and weight was determined gravimetrically. Phospholipids were removed by passing the total lipid extract through silicic acid (Bio- Sil HA, Bio-Rad Laboratories, Richmond, California) according to method of Bacon et al., (1980). Phospholipid fractions were weighed and counted by liquid scintillation. The neutral lipid fractions collected from the silicic acid columns were separated by high pressure liquid chromatography (HPLC) into cholestoryl esters (CE), triacylglycerol (TG), nonesterified fatty acids (NEFA), cholesterol (C) and diglycerides (DG) using a Lichrosorb Si-60 (25 cm x 4.6 mm, 98 10 micron pore size) column with an HP guard column (10 cm x 4.6 mm - 10 micron pore size) from Alltech Associates, Inc., Deerfield, Illinois. The methods of Bacon et al., (1982) were slightly modified. The GE and TG fraction were eluted with 6% 'methyl-t-butyl ether (MtB - Burdick and Jackson, Muskegon, Michigan) in hexane (Fisher Scientific, Fair Lawn, New Jersey) and collected separately in scintillation vials, using a model 273 ISCO fraction collector (Instrumentation Specialties Company). The NEFA and C fractions were eluted and collected by grading up the solvent polarity to 35% MtB in hexane with 1% formic acid added. Finally, the DG fraction was collected in the 100% MtB purge. All peaks for the various fractions were monitored at 210 nm using an ISCO- V4 absorbance detector. The flow rate was set at 3 ml/min (1200-1500 psi) using a Rabbit HP pump (Rainin Instrument Company, Inc., Woburn, MA). An aliquot of both the TG and NEFA fractions was assayed colorimetrically according to methods of Biggs et al.,(l975) and Novak, (1965), respectively. The remaining portions of TG and NEFA.were counted by liquid scintillation. Carbon dioxide in the blood was measured according to methods of Russel and Young, (1982) and counted by liquid scintillation. Milk fat determinations were carried out by methods of Rose-Gottlieb, (A.O.A.C., 1984) followed by liquid scintillation. Counting cocktail for nonaqueous samples was made up as 0.4% PPO and .05% POPOP in toluene. All aqueous samples were counted with the same cocktail diluted with an aqueous base. Additionally milk and urine samples were counted daily to monitor excretion of label. All counting was done on a TM Analytic scintillation counter (Elk Grove, Illinois). Calculations a) turnover (mmole min.-l) = I plateau NEFA SA 14C_ Where I is the infusion rate (uCi min.-1) of 1- palmitate and plateau NEFA SA is the mean specific activity at the asymptote, which was reached within 20-40 minutes of infusion. b) Transfer Quotient (TQ %) = SA product SA precursor x 100 Where SA of TC is expressed as dpm/umole TG fatty acid and SA CO2 is expressed as dpm/umole NEFA carbon. c) % plasma NEFA oxidized: moles oxidized day-1 (NEFA turnover, mmole min.'1) (1440 min. day-1) x 100 Where moles oxidized day -1 is on a g NEFA carbon basis. d) % of plasma NEFAeTG: = g TG from plasma NEFA NEFA turnover, g day ‘ 1x100 Pool size of respective fractions was calculated, assuming a plasma pool of 5% of body weight. 100 Statistical Analysis The data were analyzed as a split plot design evaluating treatment and period effects. Evaluations were conducted using the Genstat V statistical package (Lawes Agricultural Trust, Rothamsted Experimental Station). Individual data were also analyzed by regression in some cases and tested for significance of coefficient of correlation. RESULTS The original intent of this portion of the research was two-fold: 1) to evaluate the potential lipotrophic effect of methionine hydroxy analogue (MHA(R)), Monsanto Company, St. Louis, Missouri and, 2) simultaneously to evaluate NEFA kinetics in lactating cows in early lactation. Individual data from all the studies with lactating dairy cows is in Table 23. Thirteen individual trials were run; data obtained from the first four are limited. During the first trial, the cow collapsed after about 60 minutes of infusion for no apparent reason. The next trial, two cows were infused simultaneously and both collapsed after about 45- 60 minutes of infusion. It was determined that sodium citrate, used as an anticoagulant in the infusion and sampling cannulae, chelated enough calcium in these early post-parturient cows to cause hypocalcemia. Therefore, data from trials two and three must be interpreted carefully. These data had to be included in the MHA analysis in order to achieve sufficient degrees of freedom for testing by split plot. Exclusion of these data would have made no difference to effects of MHA. In all subsequent trials, saline was used to flush the cannulae following sampling and no further_ problems occurred. In experiment 4, after infusion, it was found that the infusion cannulae had pulled out of the vein. 101 102 ~.em m.m o~.~ o.om coo. ha.~ and saw oe.m oN mm a «cow ma m.o~ o.m~ oo.~ m.ea mos. mm.“ on New mw.~ on an o mmo~ Na o.a a.- oo.m o.m~ “no. om.e NNN mam em.m Nm on a omen an e.o~ m.aa mm.~ m.oa osu. mm.a cam woo m~.e as cm a seen on o.m o.o~ oa.m m.~ mau. em.~ man man -.m an an o swam a o.» ~.e~ om.oa o.aa woe. mm.e «an ace ao.m mm mm o mesa m o.ea o.ea mm.e m.m~ nae. um.~ Nan mom Na.m am an a seem a a.m e.aH oo.e o.o «am. do.n mam NNG -.e mm mm a omen e N.a o.- om.a N.» use. m¢.m New nae mm.m an «N a seam m --n :u- ms.m a.m nu: nu: ma“ sum so.m «N oh 0 meow e m.ma n.1m oo.m a.o oao. ca.a «we sin -n- am am a scam m ~.~a ~.~m o~.m m.aa Hum. mo.~ was «an nu- om ea c naou N we nonaeaxo N8 ea Lucas :aa\ 2: we Nuam amvex meme omen zoo .uaxm N N as on .x «Hoes .ucoo 3m Mae: ea“: .mwmum <=z um>o=u=a mammam .quA I H Houuaoo <.05. °P <.1. 108 Figure 4. Effect of stage of lactation (30, 60 and 90D) on plasma NEFA concentration. Where x is days of lactation and y is plasma NEFA concentration (uM). 109 Om .v\SOU 4 nusoo o m>8 8 o 1 >8 8 a :2 room ‘7 é 0 ¢ room fif r O O (D Icon (I_‘3M fipoq Sn'I_°1q'a‘[omn) 312.1 £13m; 113 Figure 6. Percent of NEFA turnover incorporated into TG at various plasma NEFA concentrations. y = .041 x + 27.31 (R = -.53, p‘<.1) where x is the venous concentration of NEFA (uM) and.y is NEFA incorporated into TG (Z). 114 33 <52 952.". can can can 2.: can own ow _ o o n. .n n n. D o -o. n n. rm— 0 row .3 Ton Eu 3 4 Tan >8 8 0 >8 on n re 91 6" VJBN Z 115 Figure 7. Percent of NEFA turnover oxidized to 002 at various plasma NEFA concentrations. y = .038 x + 7.79 (R = .76; p<.01) where x is the venous concentration of NEFA (uM) and y is NEFA turnover oxidized to CO2 (Z). 33 <..mz 9:85 116 TON ron Tmn ZOO ('- VJEN 34 >8 8 4 >8 8 o >8 on a DISCUSSION Increased plasma NEFA concentration are highly correlated to negative energy balance (Baird et al., 1972; Bauman and Currie, 1980). The drop in plasma NEFA concentration '(Table 26) from 30 days to 60 days is consistent with this observation suggesting lower adipose mobilization. In addition, the energy balance calculations are consistent with this observation and demonstrate that the animals were in a negative energy balance at the 30 day period. Moreover, the increases in plasma NEFA concentration are reflected in the NEFA kinetics. The significant positive correlation between NEFA turnover and concentration (Figure 5), consistent with other studies (Annison et al., 1967a; Pethick et al., 1983), suggests that tracer NEFA (palmitate) was representative of total NEFA. Similar results were obtained in other studies (Pethick et al., 1983) when oleate or stearate were used as tracers. In contrast, Annison et al., (1967a) demonstrated a poor correlation when stearate was used as a tracer. This graph also demonstrated the direct correlation between NEFA concentration and entry rate, consistent with several other reports (Annison, et al., 1967a; Jackson et al., 1968; Yamdagni and Schultz, 1969; Pethick et al., 1983) with fed and fasted ruminants. The transfer quotients reported here for TG, using this experimental method, are unique to the available 117 118 literature on ruminants. Transfer quotient (TQ) is merely on expreSsion of the percent of product arising from precursor. Because these cows were being fed during the experimental period, the exogenous TG contribution dilutes out the specific activity of the endogenous product to some extent. If the animals were fasted, then presumably there would be no exogenous contribution and at steady state the TO could reach 100% (ie. all of the TC is of endogenous—hepatic origin). These TQ values are important for determining the percent of NEFA incorporated as endogenous TG (see calculation in Methods section). The significant difference in TQ (Table 26) is not reflected in the percent NEFA incorporated as TG due to the large variation of NEFA turnover values. In addition, two assumptions were made for the calculation of percent NEFA incorporated into TG, 1) a plasma TG concentration of 20 mg/dl was assumed, due to problems experienced in HPLC TG separation, and, 2) direct measurements of TG half-life could not be made from the data and a TG half-life of 4.5 min. for plasma TG was assumed from Glascock and Welch, (1974). Obviously, the extent to which these assumed values varied would affect individual calculated responses. Two observations are noteworthy from the calculated figures for percent NEFA converted to TG (Table 25). First of all, the low values (9% and 18%) suggest low endogenous production of TG from the precursor plasma NEFA pool. Sesandly, the percent NEFA incorporated to TC is somewhat attenuated at 30 days compared to 60 days (9% vs. 18%, respectively). Previous studies 119 attempting to evaluate endogenous versus exogenous contribution to milk fat TG by measuring transfer of labelled fatty acid into milk are consistent with these results (Palmquist and Mattos, 1978). These investigators suggested exogenous contribution to milk fat to be 85% to 90%, with endogenous contributing very little (10% to 15%). If a value of 15% (NEFA turnover converted to TC) is assumed, then given a daily NEFA turnover of 3.816 moles/day, a total of 572 mmoles/day (3.816 moles/day x .15 = 572 mmoles/day) of fatty acid is converted to TG. Assuming 3 moles of fatty acid per mole of TG, this gives rise to a potential for 191 mmoles of TG per day. A 500 kg cow producing 30 kg of milk/day at 3.5% fat which is 90% TG and further assuming 50% of the milk TC is from.plasma TG hydrolysis and 50% from ”de novo" synthesis in mammary gland (Palmquist et al., 1969), means that about 555 mmoles of TG are required from plasma. This also assumes that all plasma TC is utilized solely by the mammary gland. Just over 30% of this (191 mmoles/555 mmoles - 34%) could be of endogenous origin, the rest would have to be of dietary origin or direct NEFA uptake. The calculated value of 34% would be even less (about 23%) at day 30 of lactation. These values are inconsistent with the view that hepatic VLDL are primary contributors to milk fat in early lactation (Puppione, 1983) and in fact suggest that hepatic contribution is diminished in early lactation. Moreover, the negative correlation between plasma NEFA concentration and percent of NEFA turnover converted to TG suggests that increases in plasma 120 NEFA do not provide proportional increases in substrate for TG synthesis and secretion (Figure 6). In support of the above results, the sudden mobilization of NEFA from adipose that occurs post partum appears to initiate varying degrees of fatty liver in most cattle with recovery occurring by about 8 weeks (Gerloff, 1985). Field studies have suggested that about one-third of all peripartum dairy cows develOp moderate to severe hepatic lipidosis (HL) while the rest develop more mild forms (Reid and Roberts, 1983 and Gerloff, 1985). Development of HL has been associated with decreases in hepatic output of TG (Reid et al., 1979; Herdt et al., 1983). The development of HL is consistent with the theory that the post partum mobilization of fatty acid and subsequent formation of hepatic VLDL may in fact be negatively correlated. If fatty acid mobilization and formation of hepatic VLDL are negatively correlated, then direct uptake of NEFA along with diet become the major plasma lipid substrate for the mammary gland. In fact, studies have shown significant mammary uptake of NEFA in the cow (40 qu/L to 200 qu/L) when NEFA concentration reached 300 qu/L to 750 qu/L (Kronfeld, 1965). Small and variable NEFA arterio-venous differences have been detected in fed goats (Barry et al., 1963; Annison et al., 1967b). Studies utilizing a combination of infused radiolabelled NEFA and mammary arterio-venous concentration differences in combination with blood flow measurements have demonstrated substantial uptake of labelled NEFA (Annison et al., 1967b). The uptake of labelled NEFA was reflected both in transfer of 121 label into milk fat and in a drop in NEFA specific activity across the mammary gland. However, results of mammary arterio- venous differences in combination with blood flow in the same study, suggest very little net uptake of NEFA by the mammary gland. The difficulty in detecting NEFA uptake may be due to lipoprotein lipase (LPL) activity on blood TG with consequent release of fatty acids from TG which dilute out the specific activity in the mammary vein. Small arterio-venous differences could be substantial in view of the increased blood flow to the mammary gland during lactation. For example, a net NEFA difference of 80 qu/L, assuming a blood flow of 14L/min (x - 30 kg/day milk production; y = .55 + .44 x; Kronfeld et al., 1968) would yield enough preformed plasma NEFA to provide substrate for milk fat TG (using previously calculated requirements of 555 mmoles of TG). The positive correlation between plasma NEFA concentration and milk fat production (Figure 3) also suggests direct uptake of NEFA by the mammary gland. Another pertinent point in this discussion regards the TG half-life assumed in the calculation of percent NEFA converted to TG. The value of 4.5 minutes (Glascock and 3H_ Welch, 1974) was determined by an intraruminal dose of palmitic acid and therefore was representative of intestinal VLDL half-life. Another study, reporting half-lives of 1.5 minutes to 2.5 minutes, utilized radiolabelled lipoproteins harvested from calf lymph which were reinjected into lactating cows (Palmquist and Mattos, 1978). No studies with 122 ruminants have determined the half—life of the endogenous VLDL specifically. Indications in other species are that hepatic and intestinal VLDL are metabolized at different rates, with the half-life of hepatic VLDL being much slower (Grundy and Mok, 1976). The possibility of a slower half-life for endogenous TC is also suggested by our data of plasma TG specific activity curves (Figure 8). Low activity is seen after 20 minutes, rising to a peak at 80-120 minutes in most cases. The infusion is stOpped at 160 minutes and the curve of TG specific activity remains relatively flat out to 250 minutes. If the half-life of the endogenous TG were around 2 to 5 minutes, the curves should have dropped much faster. A single sample taken 18 hours later, detected considerable plasma TG specific activity still present. Obviously, if the half-life of endogenous TC is slower (i.e. longer than 4.5 minutes) it will be reflected in even lower amounts of NEFA converted to TG. To avoid repetition, a discussion of this curve and its interpretation will be elaborated on in the next section of the ewe data in the experiments with wethers. Finally, the data on palmitate oxidation suggest that percent of CO2 from NEFA oxidation and total NEFA oxidized were lower at 60 days compared to 30 days of lactation (Table 26). This is further demonstrated by the graph of plasma NEFA concentration (uM) versus percent NEFA oxidized to CO2 (Figure 7). A correlation of .76 demonstrated a significant and direct relationship between plasma 123 Figure 8. Plasma specific activity of NEFA (dpm/nmole) and TG (dpm/umole TGFA) in MHA XI where x is time (minutes) and y is specific activity (S.A. dpm/umole). $33.25 2:? oan anal omm - 2.8 . etc. + WW— . 2.; (ow. - ow: (We 8 9 on o BI dag. . - » - r o. $.sz i a row i T h .8. 4’ 2 1 it 1 moon 1 wooe— c rooon (clown/map) 'V '3 125 NEFA concentration and percent NEFA oxidized to C02. These results are similar to those obtained with fed and fasted pregnant ewes (Pethick et al., 1983) and lactating cows (Jackson et al., 1968). Other studies in the fed lactating goat (Annison et al., 1967a) and fed and starved sheep (Annison et al., 1967b) have demonstrated similar transfer quotients contributing from 1% to 5% of total C02. Based on these results, it appears that the stress of lactation and subsequent mobilization of NEFA from adipose, provides a significant contribution to the energy requirements of the lactating cow; In addition, the reciprocal relationship between percent NEFA turnover oxidized to CO2 (Figure 7) or converted to TG (Figure 6) demonstrated by these studies seems to reflect a metabolic transition generally seen in times of physiological stress, e.g. fasting or experimental diabetes in rats (Heimberg et al., 1974, Mayes, 1970), which allows the body to adjust to a variety of substrates to support necessary metabolic processes. ENDOGENOUS TURNOVER OF HEPATIC AND PLASMA TRIACYLGLYCEROL IN SHEEP 126 MATERIALS AND METHODS Two studies were conducted with sheep obtained from the M.S.U. sheep barn. The first study evaluated endogenous TG production by intestinal lymph using two mature crossbred nonpregnant ewes. The second study was designed to evaluate endogenous TG turnover in liver and plasma of 4 Correidale crossbred wethers. Ewe study - endogenous intestinal TG production. The ewes were pastured on a brome/bluegrass mix and were maintained in a fed state until surgery. The animals were transported to the M.S.U. Veterinary Clinic the afternoon before the experiment. The morning of the experiment the ewes were weighed and the lateral para lumbar fossa area extending cranially to the tenth rib was clipped and scrubbed for surgery. Following an intravenous injection of Thiopental (2.5%), the ewes were intubated and attached to an inhalation anaesthetic (2.5% Halothane) for the remainder of the experiment. An incision was made parallel to the last rib extending downward around the rib cage about 30 cm. The surgical procedure of Katz and Bergman, (1969a) was followed h(R) catheters, 16 g with minor modifications. Angiocat (Deseret Medical, Inc., Sandy, Utah) were used to cannulate the mesenteric and portal veins. An 18 g polyvinyl chloride 127 128 (PVC) catheter that had been immersed in 2% TDMAC-heparin (Polysciences, Inc., Warrington, PA) was used to cannulate the intestinal lymph duct (Lascelles and Morris, 1960). Following catheter placement and experimental procedure (usually about 5 to 6 hours), the ewe was euthanized with sodium pentabarbital (8%) and taken to the Veterinary Necropsy Laboratory to again verify catheter placement. The carcass was disposed of by incineration. Immediately following catheter placement, a 100 uCi 1l‘C-palmitate was injected into the mesenteric dose of l— vein cannula. Approximately 15 ml of blood was sampled from the portal vein cannula into tubes containing EDTA (lmg/ml) and protamine sulfate (.5mg/ml). Sampling times twere 0, 2, 4, 6, 8, 10, 15, 20 minutes and every 10 minutes thereafter to 100 minutes, followed by 20 minute samples to 180 minutes. Intestinal lymph was drained into tubes containing EDTA (1 mg/ml) in 15 minute fraction collections up to 180 minutes. Lymph blood and tissue samples were all kept on ice and transported back to the lab for analysis. All blood and lymph samples were centifuged in a Damon IEC Model K centrifuge (Needham Heights, Mass.) at 2000 xg for 20 mdnutes and plasma was drawn off and stored at -60°C until it was extracted. Wether study - endogenous TG turnover. The wethers, obtained from the M.S.U. sheep barns, were transported to the M.S.U. Veterinary Clinic the day before the experiment. The animals were weighed and the 129 same area of the animal was prepared as described earlier for the ewe study. A jugular vein catheter was inserted 14C-palmitate and for introduction of a 200 uCi dose of 1- subsequent blood sampling at 0, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 100, 130, 160, 200, 250, 300, 360, 420, 600, 840, 1200, 1440 and 1680 minutes post injection of dose. Blood samples were handled as previously described in the ewe study. A local anesthetic of isoprOpanol:Lidocaine (50:50) was given at the incision site (posterior to the last rib) and liver tissue was biopsied at 15, 30, 60, 180, 360, 600 and 1680 minutes post injection of dose. All liver samples were placed in dry ice immediately and frozen for analysis two days later. Tissue was biopsied with a modified 3 ml plastic syringe (cutting off the syringe tip end) by coring and applying light suction. A 0.5 to 1.0 g sample of liver was consistently obtained. Following biopsy, Gel Foam: e .5 E 00._. 00.0 00.0 0¢.0 0N0 00.0 r L h [— L L l— O IN 0 I 6 11V I r 10 I I 10 a IOP _oEomotBE I 1 0630.6 L8 0 143 Results of the plasma TG kinetics are shown in Table 29. Pool sizes were quite variable with actual plasma TG concentrations ranging from 5 mg/dl to 20 mg/dl between animals. Concentrations were, however, fairly steady throughout the period of the experiment as evidenced by the standard errors. Variation in pool size necessarily affected turnover rates which ranged from 6.5 to 42.1 mg.h:1 kg bwt'l. The half life of plasma TG averaged 10 minutes in the four experiments. Plasma NEFA concentration was quite variable between animals but appeared to remain stable throughout the experimental period (Table 30). Finally, Table 31 shows the results of peeling the fast (microsomal) and slow (fat droplet) curves and the respective turnover rates and pool sizes. Pool sizes, determined directly, were variable between animals, but consistent with the differences in % liver lipid and % liver TG reported in Table 28. Pool sizes for the slow pool are the result of combining the cell debris TG and fat droplet TG pools as both S.A. curves in the experiments were very similar; Half lives averaged 41 minutes‘and 23 h, respectively for the fast and slow pools. Also of interest are the correlations between liver TG percent and turnover rates of the fast (microsomal pool - R =186) and slow (fat droplet - R = .99) pools (Figure 11). 144 .Hm>HH anu GHnuHs maoom .m.m H cmaEn uafiaouv any can HmsomouoHE ozu ou Human 30am 0cm ummmm cm. H 0N.N H000.H meooo. 500 H 0<¢m 0.“ H 00.5 mq00. H mfio. ma H 05m 0.H H 5.00 m 00.8 m000. «on H 0000 No.0 “#0. cm H 0~¢ 0.00 00H 00. N000. ham H wwm~ 00.- . 0N0. 0N H NON m.~¢ ~00 ~q.~ 0000. 0mm H “emu ~0.s wfio. «N H 0~m m.me 0N0 «0.0 5000. £0~¢ H 000m -.m 000. now H 050 0.00 050 .GHE we .u3n m 5.05 .cHE we as o w on“ I I. .I I. auaaa uu_-; a L we maaa Hooa a and a 8x «mam Hoom use a «Sofim munch we au>aa museum: ca monumcae up am>aa .Hm magma DISCUSSION The difficulties inherent in studies of this type lie primarily in the ability to sample tissues with a minimum of trauma so as not to disturb steady state. Isotope studies assume steady state conditions. The extent to which the system varies from steady state will influence the outcome of the calculations. Endogenous Intestinal Lymph Production. The studies conducted in the mature pregnant ewe lasted 3 hours and steady state conditions were observed. Over this time period, endogenous TG production from the intestine did not contribute significant activity to the total plasma TG pool (Table 27). These results are consistent with studies conducted in rats (Baxter, 1966). One study in a hepatec- tomized dog (HavelandGoldfien, 1961), demonstrated similar TG specific activity in thoracic duct lymph compared to plasma. This may have resulted from either direct transfer of labelled TG or esterification of capillary filtrate NEFA. Although capillary filtrate of blood lipid into lymph has been shown to occur (Leat and Hall, 1968), demonstrative evidence of NEFA esterification to TG is lacking and therefore endogenous intestinal TG contribution was assumed to be negligible and unlikely to influence measurements of endogenous TG turnover. 145 146 Endogenous TG Turnover Study Biopsy methods were successful and .5 to 1.0 g of tissue was obtained consistently from 4 wethers. The variation in NEFA concentrations between experiments over the 28 h period (Table 30) are suggestive of varying degrees of stress in response to the surgical methods. The animals did move about and were observed to urinate, defecate and drink water during the 28 h period. Feed intake was diminished but did not appear to affect steady state as evidenced by the average plasma and liver TG concentrations (Tables 29 and 31) and plasma NEFA concentration (Table 30). The general method for determining TG turnover has been validated and described previously in studies with human subjects (Farquhar et al., 1965). The solution can be determined graphically by interpolation of the TG S.A. curve back to the y-intercept and determining the slope (K). Alternatively, the use of assumptions of plasma percent of body weight, and measured plasma concentration of TG to determine pool size combined with a determination of slope (K) are also valid. The use of the above method for species other than humans and perhaps avians has recently been questioned (Baker, 1984). Methods used to validate this approach involve preparation of endogenous lipoprotein by injection of labelled precursor and sampling of large quantities of blood at tmax of the TG S.A. curve. Subsequent isolation of plasma VLDL and reinjection into another host should generate a curve similar to the one generated by 147 injection of the endogenous NEFA. Reinjection of labelled VLDL in dogs (Gross et al., 1967), pigs (Hannan et al., 1980), rats (Baker and Schotz, 1964) and rabbits (Havel et al., 1962) suggest that degradation is much faster than would appear from measurement of the curve generated by injection of labelled precursor. Half-lives of 15-25 minutes measured with VLDL reinjection are compared to 1-3 hours measured by labelled precursor injection. Similar studies carried out in human subjects showed that reinjection of VLDL generated a curve similar to injection of a labelled precursor (Farquhar et al., 1965). Other studies in human subjects are at variance with this (Havel and Kane, 1975). These investigators utilized VLDL that were isolated and reinjected within 2 hours and results were similar to those reported for dogs, pigs, rats and rabbits (see above). Havel and Kane, (1975) suggested the length of time and storage conditions (4 days, 4°C) in the study of Farquhar may have caused changes in the composition of the lipoproteins. Isolation of bovine lipoproteins below 20°C results in more TG in the more dense intermediate lipoprotein fraction due to changes in VLDL composition (Grummer et al., 1986). Although VLDL compositional changes may not occur as rapidly in other species, perhaps after prolonged storage at these low temperatures, some changes may occur. .Indeed, TG removal from plasma appears to be a function of size due to TG content (Streja, 1979). Therefore, compositional changes may affect removal rate in the VLDL reinjection studies. The responses seen in the non-human species following reinjection of VLDL cannot be explained on this basis, however, as they 148 were also held for 4 days at 4°C before being reinjected (Gross et al., 1967). Baker, (1984) further suggests that the differences between humans and other species seen as a result of the VLDL reinjection validation procedure (disregarding results of Havel and Kane, 1975) are a reflection of the turnover of the hepatic TG compartment. In fact, studies in dogs (Cross at al., 1967) and pigs (Hannan et al., 1980) demonstrate that the plasma TG S.A. curve following injection of labelled precursor mirrors the fall in hepatic TG S.A. in some of the more rapidly turning over TG compartments, including the rough and smooth endoplasmic reticulum and Golgi apparatus. Baker, (1984) suggests that in these species the plasma VLDL-TC compartment has a more rapid turnover and has very little influence on the falling portion of the curve. Moreover, the early rising part of the curve (Ka) should represent primarily the fractional rate constant of the plasma VLDL-TC compartment, provided the latter is faster than its precursor; which seems to be the case with the non-human species. This would be the reverse in humalsubjects and species in which the liver precursor pool turns over faster than the plasma VLDL-TC compartment. Similar reinjection studies were not carried out in the wethers in this study. However, scrutiny of the kinetic curves for the hepatic microsomal pool and the plasma TG pool (Figure 10) demonstrate homologous rates of decay. In fact, calculation of half life from the hepatic microsomal curves in all experiments average 42 minutes compared to about 149 41 minutes using the plasma curve. The results of the ip_yip£p studies in an earlier chapter also support the limiting role of the liver in hepatic TG secretion in ruminant species. The slowly degrading portion of the curve has been shown to be a reflection of a slowly turning over pool of TC within the liver (Zech et al., 1979 and Shames et al., 1970) and appears in plasma only after about 12 hours (human studies). In the current studies, it was observed between 5 and 6 hours, (Figure 10). Shames et al., (1970) showed that the recycling of a large fraction of TG fatty acid back to the liver as NEFA could account for only a small fraction of the observed "tail" of the S.A. time curve for TG fatty acid in VLDL. Most of the current modelling of hepatic TG production includes a substantial pool of relatively inert TG through which a large portion of the label may pass (Zech et al., 1979; Shames et al., 1970; Quarfordt et al., 1970). Kinetic evidence for a large hepatic pool of TG having a low S.A. has been reported in dogs (Gross et al., 1967), rabbits (Havel et al., 1962) and rats (Stein and Shapiro, 1959). Results of the present studies in wethers also suggest a large fairly inert hepatic pool of TG. Direct measurements of pool size in wethers suggest that inert "fat droplet" pool may be 5 to 10 times larger than the more rapidly turning over "microsomal" pool (Table 28). Similar results were not observed in pigs, however, in which there seemed to be a relatively hommgenous precursor pool for lipoprotein production (Hannan et al., 1980). 150 Subsequent calculations of turnover rates for the fast "microsomal” pool, ranging from 5 to 12 mg.h-l kg bwt-} (Table 31), are similar to results observed in Triton injected goats (Schultz and Esdale, 1971) where rate of 1 entry into plasma was measured to be from 8 to 13 mg h-. kg bwt-} in the fed state. The slow turnover rate of the fat droplet pool (Table 31) and the correlations observed in Figure 11 lend support to current theory on ruminant hepatic TG metabolism. The negative correlation (R='?86) between turnover rate in the fast pool and % TG in the liver, suggest that as hepatic TG % is increased due to increased uptake of blood NEFA, hepatic TG secretion is decreased. This is consistent with previous studies in dairy cattle (Herdt et al., 1983), and is consistent with decreases in TG transfer quotients observed in early lactating cows in the Ohio study previously mentioned in this thesis. The positive correlation (R= .99) between turnover rate in the slow pool and % hepatic TG may be reflecting the diversion of esterified fatty acid to other intrahepatic metabolic pathways such as oxidation and ketone body production with subsequent decreases in TG secretion. The model depicted in Figure 12 is similar to that of Gross et al., (1967) and Hannan et al., (1980). Pool sizes and turnover rates between compartments are mean values from the four experiments with wethers. The possibility of recycling from the plasma TG compartment to the precursor hepatic TG pool is depicted by the dashed line. The arrow 151 1:23.22 . Plasma Fat Droplet 7 7 ..- - Intrahepati Metabolism 4.2.3 .A I i ' 12.2 __°' Microsomal delayl 7 '8 . 20 trahepatic 8.4 . 1 I etabolism Figure 12. A tentative model for endogenous TC turnover in the liver and plasma of the wether. Numbers in boxes indicate pool size (mg TC per kg bwt.) Turnover rates are indicated by arrows expressed as mg TC h’1.kg bwt ’1. Values are means of data from four wethers. Liver fat droplet and microsomal pools were determined by direct measurement. The input from the outside into the plasma pool is assumed to be intestinal contribution to plasma TG. This value was calculated as the difference between measured rates of plasma TC turnover and that of hepatic TC turnover. The A 2.3 in the inert pool is the turnover rate mg.h'k kg bwt '1 in the fat droplet pool Dashed lines are indicative of potential pathways not'defined within this experiment. 152 entering the plasma pool is representative of intestinal TC contribution and was calculated as the difference between plasma TC turnover and the hepatic "fast" pool turnover. The 43 2.3 in the inert pool depicts turnover which may occur between the microsomal, mitochondrial and other intrahepatic pools. 1. kg bwt-l Finally, an entry rate of 7.8 mg.h- entering plasma from the hepatic fast pool in wethers compares to values in pigs of 12.3 (Hannan et al., 1980), dogs, 10.8 (Gross et al., 1967), rats, 156 (Baker and Schotz, 1964), and humans, 240 (Farquhar et al., 1965) mg.h-1.kg bwt.-1' The lower turnover rates mentioned in dogs, pigs and here in sheep, support the limiting role of the liver in TC production in these species. SUMMARY AND CONCLUSIONS Secretion of TC from ruminant liver slices was very low and in most cases no significant difference was observed between 4 hour and 8 hour incubation periods. Attempts to stimulate TG secretion through the addition of various hormones and substrates to the media were unsuccessful. A decreased time interval from slaughter to incubation did not appear to improve secretion rates. The preincubation tissue holding temperature (room temperature or ice) did not affect secretion rates. Neither type nor level of fatty acid in the media was effective in stimulating secretion of TC. A comparison of Krebs Ringer buffer media (KRB) and Minimal Essential Media (MEM) with Earle's salts and 25 mM Hepes buffer demonstrated greater rates of tissue esterification with MEM but did not affect secretion. Addition of serum or propionate failed to increase TC secretion. TC secretion in a young Holstein calf (milk fed) and a neonate crossbred lamb were also low, comparable to adult ruminants. In contrast to the ruminant, TC secretion from rat liver slices was linear over the period of incubation (0-8 hours). Secretion of TC measured in the rat appeared to be a reflection of VLDL-TC secretion as puromycin, a known protein inhibitor, greatly diminished TG secretion. A consistent species difference in TC secretion rates was also observed. In species in which the liver is the major site of fatty acid 153 154 synthesis (chicken and fish) secretion rates were the highest. In contrast, in species where hepatic fatty acid synthesis is limited (cattle, sheep, pig and guinea pig) secretion was also limited. Moreover, the rat and rabbit, which fall in between the two extremes in terms of hepatic fatty acid synthesis, secreted TC at rates midway between the two extreme species groups. This observation did not hold for rates of esterification. Rates of esterification although variable did not suggest a similar species difference. Results of these studies demonstrate a species difference in the ability to secrete TC from.the liver as lipoprotein. Moreover, the ability to secrete TC appears to be related to the capacity for hepatic fatty acid synthesis. The results of the ip_yiyp studies in sheep and lactating cows demonstrate that the ruminant liver does secrete TG. However, the hepatic TC model derived from the wether data, (Figure 10) and Baker's, (1984) interpretations of endogenous TC kinetic studies, suggest that the liver TG compartment turns over very slowly. The slow hepatic turnover rates measured in dogs (Gross et al., 1967) and pigs (Hannan et al., 1980) are consistent with low production rates from liver slices of species that have limited hepatic fatty- acid synthesis. In addition, the observation that in humans and avian species the plasma TC turnover is rate limiting and therefore that liver production rates are much faster (Baker, 1984), is consistent with the ip|yip£p TC secretion rates measured in chickens in this study and also with the 155 liver's role in hepatic fatty acid synthesis in these species. The results of the present studies in the lactating cow are also consistent with this line of reasoning. The increase in precursor NEFA mobilized from adipose in early lactation did not appear to generate proportional increases in product TC. In fact, calculations of the potential contribution of endogenous TG to the mammary gland for milk fat production suggest that endogenous TC production could account for about 20% of what is required at 30 days increasing to about 35% at 60 days. These calculations also assume that all endogenous TC production is diverted only to the mammary gland. In addition, the rapid transfer of labelled fatty acid into milk fat seen in this study also suggests that direct uptake of plasma NEFA may make a significant contribution to milk fat production, especially in early lactation. A slow turnover of hepatic TC is also consistent with theories on the etiology of hepatic lipidosis in ruminants. The liver is capable of extracting and esterifying the plasma NEFA to TC but appears to metabolize that TC very slowly. The relationship between hepatic fatty acid synthesis and secretion of VLDL-TC is not well understood. From a physiological standpoint, the relationship between hepatic fatty acid synthesis and secretion as TC makes sense. If lipogenesis is limiting in the liver under normal conditions, than the liver does not have to handle excesses of lipid and would not need an active secretion mechanism. In the 156 case of ruminants, the potential competition between simultaneous hepatic gluconeogenesis and lipogenesis for ATP and carbon skeletons and the importance of acetate as an extrahepatic energy source are good reasons for limited hepatic lipogenic activity. A better understanding of specific factors regulating formation and secretion of hepatic VLDL is needed. Results of the present study suggest that some factor or factors related to hepatic lipogenic capacity are required for VLDL secretion. In general, the species difference in TC secretion appears to reflect the metabolic state of the liver. In rats, TG secretion is greatly diminished in the fasted state even in the presence of high levels of preformed fatty acid. The liver of the ruminant even in the fed state, appears to reflect that of the fasted rat in which limited lipogenic activity occurs. APPENDICIES 157 Appendix 1 Composition of Minimal Essential Media (MEM)1 with Earle's Salts and 25 mM Hepes Buffer and without L-glutamine. Components Inorganic salts: CaClz(anhyd) KCl MgSoa.7H20 NaCl NaHCO3 NaH2P04.H20 Amino Acids L-Arginine.HCl L-Cystine Liquid (1x) mg/L 200 400 200 6800 2200 140. 126 24 L-Histidine HCl. H20 L-Isoleucine L-Leucine L-Lysine HCL L-Methionine L-Phenylalanine L-Threonine l 42 52 52 72. 15 32 48 00 Comppnents con't. Amino Acids L-Tryptophane L-Tryosine L-Valine Vitamins D-Ca-pantothenate Choline chloride Folic Acid i-inositol Nicotinamide Pyridoxal-HCl Riboflavin Thiamine-HCL Other D-Glucose Phenol Red Liquid (1x) mg/L 10 36 46 l—INH l-‘l—I 1000 10 GIBCO LaboratOries, Grand Island, NY (Cat. No. 380-2360). 158 Appendix 2 Media Extraction Procedure lp_Vitro Assay 1). 2). 3). 4). 5). 6). Two m1 of media are extracted with 9 ml of 3.2 (Hexane: Isopropanol) in 16 x 150 mm screw cap test tubes. Vortex 30 seconds. Add 7 ml 6.7% NaZSO4 and vortex 30 seconds with cap on. Centrifuge full speed in clinical centrifuge (Damon, IEC Model K centrifuge, Needham.Heights, MA) for 5 minutes. Pipette supernatant into clean 16 x 150 mm screw cap tubes with 1.6 gms. activated alumina* (Triglyceride Purifer, Sigma Chemical, St. Louis, Missouri). Wash meniscus 2 x with 2 ml 7:2 (H:I). Place capped tubes in test tube rack inside metabolic shaker (Dubnoff, Precision Scientific, Chicago, Illinois). in horizontal position. Shake for 15 minutes. Shake alumina off sides and centrifuge full speed for 5 minutes. Pour off supernatant into 20 ml test tube. Add 4‘ml of 7:2 H:I to activated alumina, vortex, centrifuge and add to supernatant in 20 ml test tube. Evaporate extract and transfer to 12 x 75 mm test tubes (capped). Store until used for TLC spotting. *activated alumina step chelates labelled NEFA decreasing contamination of TG spot during TLC. 159 Appendix 3 Summary of HPLC procedure The sample resuspended in an appropriate amount of 2% methyl-t-butyl ether (MtB) in hexane is injected into the column on 2% MtB (flow rate 3ml/min). At 3ml the solvent gradient is increased to 8% MtB in hexane and the 0-9ml fraction collection contains cholesterol ester (CE). The 9-24ml collection, eluted by 8% MtB, contains the triacyl- glycerol (TG). At 18ml a gradient increase to 35% MtB in hexane (with 1% formic acid added) will elute the non- esterified fatty acid (NEFA) fraction in the 24-36ml collection. At 30ml the gradient is increased to 100% MtB and the column is cleaned off. Finally at 48ml the gradient is decreased to 2% and allowed to drop to baseline in preparation for the next injection. An average separation requires 25-30 minutes. 160 Appendix 4 Composition of Krebs-Ringer Buffer (KRB) and Minimal Essential Media (MEM) . MEM, KRB 1110 <_mM_> Acetate --- 2.5 Propionate 1.0 1.0 Glucose 5.0 2.2 Carnitine --- 2.0 Palmitate .2 .2 BSA .05 .05 BIBLIOGRAPHY BIBLIOGRAPHY Abumrad, N. A., J. H. Park and C. E. Park, (1984). Permeation of long chain fatty acid into adipocytes. Kinetics, specificity and evidence for involvement of a membrane protein. J. Biol. Chem. 259:8945. Abumrad, N. A., R. C. Perkins, J. H. Park and C. R. Park, (1981). Mechanism of long chain fatty acid permeation in the isolated adipocyte. J. Biol. Chem. 256:9183. Alexander, C. A., R. L. Hamilton and R. J. Havel, (1976). Subcellular localization of B apoprotein of plasma lipoproteins in rat liver. J. Cell. Biol. 69:241. Annison, E. F., R. E. Brown, R. A. Leng, D. B. Lindsay and C. E. West, (1967a). 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