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I:J'L'i¢'1‘lr:1'v‘ll’mr(;§ ,{fi'i'zi'f-‘v'l‘i‘ V :i;: 1' 1‘1 , . ::.~:.,'1;.r;1'. “I" $353315; “:“1‘ 1 1'3; A} 3,: A 15",, 1 E" uin‘ififq‘ If?! 1" r 1 f" 1554'!“ 1,: Ar}; 31311;: '3); if?“ ‘ 1’1"“va ‘ ftfl-w (f ’F (‘21.: ' ;:{;;1' ;:'. AS‘C‘v £;;[ ‘ i”“§{é"~"y{k"fi ‘l {L :I’J‘l ‘14,; “1 .( ‘1‘??? l‘ “:(O ‘fitga ‘gétgw LIBRARY Mich gan Sta te Un Nersity o This is torcertif t tth'e . " . y- gum-£221 thesis entitled » latory Aspects Of Triglyceride Uptake if .7 y Bovine Adipose and Mammary Tissues presented by John E. Shirley , g has been accepted towards fulfillment of the requirements for M—degree inDairx Science and Institute of Nutrihon fly //5MW ajor professor Date /.3 467M/f/77j _ (/ 0-7639 4" ..-~ i—fl ‘_..-. ,____________“____, .— .___ “+3— ABSTRACT REGULATORY ASPECTS OF TRIGLYCERIDE UPTAKE BY BOVINE ADIPOSE AND MAMMARY TISSUES By John E. Shirley Lipoprotein lipase (LPL) and glyceride synthesis (GS) are considered essential for uptake of blood triglyceride fatty acids by extrahepatic tissues. Two experiments investigated the regulatory capability of LPL and GS in bovine adipose and mammary tissues. Ini- tially (Experiment T1) samples of adipose and mammary tissues were obtained from nine primiparous Holstein heifers l80 days (n = 6) or 260 days (n = 3) in gestation and l4 days after induced (caesarean section) lactation. In the second experiment (T2) biopsy samples of mammary and shoulder subcutaneous adipose tissue were taken at 49, 21, 14, 8, and 2 days prepartum and 7, l4, 28, 60, 120, 180, 240, and 300 days postpartum from eight multiparous Holstein cows. Mammary LPL activity (umoles fatty acids released hr'1 10 mg tissue protein-1) increased from .5 to 46.9 while adipose LPL de- creased (12 to 8) with onset of lactation (Experiment T1). Similarly, GS activity (umoles palmitate incorporated hr'1 10 mg tissue protein-1) l John E. Shirley increased 8 fold in mammary tissue and decreased 6 fold in adipose tissue. In Experiment T2, mammary LPL activity (umoles glycerol re- leased hr.1 100 mg tissue protein-1) increased 6 fold between 49 and 7 days prepartum, then increased sharply (.4 to 5.1) between 7 and 2 days prepartum, reached a maximum (83 i 12) at 120 days postpartum, and then decreased slowly until 280 days postpartum (19 i 18). Mammary GS activity (umoles palmitate incorporated hr-1 100 mg tissue protein-1) decreased slightly between 49 and 8 days prepartum then increased 5 fold by 2 days prepartum and an additional 2 fold by 2 weeks postpartum after which it remained relatively Constant until 280 days postpartum. Adipose tissue LPL and GS activities appeared to be more sensitive to ~fienergy status than to changes in lactational state (Experiment T2). Although activities of the adipose enzymes were variable, two discern- ible peaks occurred; one 2 days prior to parturition and one during mid-lactation (120 to 180 days). Both peaks correspond to luxury grain intake. Availability of LPL for release into plasma may represent up- take of triglyceride fatty acids by a tissue better than total tissue LPL activity since LPL catalyzes hydrolysis of blood triglyceride at the capillary membrane.‘ Four experiments were conducted to ascertain the feasibility of using mammary venous plasma lipolytic activity (PLA), against a substrate of triolein emulsion activated with serum, 2 John E. Shirley as a measure of mammary clearance of triglycerides from blood. Pre- heparin and peak-postheparin PLA were measured and compared to milk fat production (an overt measure of plasma triglyceride utilization by the mammary gland). In subsequent experiments, PLA was characterized with respect to: 1) LPL and GS activities in adipose and mammary tissues, 2) energy status of the cow, 3) milking stimulus, and 4) changes in lactational state. Samples were taken simultaneously from the jugular and mammary veins and the difference in PLA between the two (M-J) was assumed to be the mammary gland contribution. Preheparin mammary PLA (M-J) was: 1) positively correlated .8, p < .05) with milk fat production, mammary tiSsue LPL activity A us ll .7, p < .01) and mammary tissue GS activity (r = .6, p < .05); A .1 ll 2) negatively correlated with energy status of the cow (r = —.3), adipose tissue LPL activity (r = -.7, p < .01) and adipose GS activity (r = -.3); and 3) sensitive to mammary gland emptying and prolactin injection (i.v.). Postheparin PLA in mammary venous plasma (M-J) was negatively correlated (r = -.4) with milk fat production and positively correlated with energy status of the cow (r = .7). Preheparin PLA was not detectable in either jugular or mammary venous plasma of primiparous heifers at 17 or 14 days prepartum, but was detectable between 8 and 2 days prepartum. Mammary PLA (M-J) in- creased significantly (p < .01) on day of parturition and attained a 3 John E. Shirley maximum by 2 to 4 days postpartum. Postpartum PLA (M-J) was signifi- cantly higher (p < .01) than PLA prepartum or PLA on the day of par- turition. No difference in heparin releasable PLA between jugular and mammary venous plasma was observed before 7 days prepartum. A mean positive M-J difference in postheparin PLA occurred by 4 days prepartum. Postheparin PLA in mammary, jugular, and mammary minus jugular venous plasma increased sharply at parturition followed by an additional in- crease by 2 to 4 days postpartum. Results of these experiments are consistent with the views that (1) the redistribution of lipid from adipose to mammary tissue at in- duction of lactation is due to a decrease in the uptake ability of adipose tissue and an increase in uptake ability of the mammary gland, (2) LPL released to mammary plasma is a more plausible regulator of triglyceride fatty acid uptake by the mammary gland than GS, (3) pre- heparin PLA in mammary venous plasma reflects the mammary glands abil- ity to clear triglycerides from blood more accurately than tissue LPL activity or postheparin PLA, and (4) adipose tissue LPL and GS are more sensitive to energy status than lactational state. Results numbers 2 and 3 represent important new concepts. REGULATORY ASPECTS OF TRIGLYCERIDE UPTAKE BY BOVINE ADIPOSE AND MAMMARY TISSUES By John E..Shir1ey A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy and Institute of Nutrition 1973 Dedicated: To my parents who instilled in me a thirst for knowledge and the value of a smile and a funny story. ii ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to the following: Dr. Roy S. Emery for his inspiration, patience, and valuable counsel during the course of this study and for his help in the preparation of this thesis. Dr. Edward Convey, Dr. Evelyn Rivera, Dr. Rachael Schemmel, and Dr. William Wells for serving on my graduate committee and for criti- cally reading this manuscript. Dr. David Morrow, Dr. Wayne Oxender, and the veterinary students for their assistance in obtaining tissues by surgical biopsy. Mr. Rodney K. McGuffey for his technical assistance and friendship. Mr. Valden Smith for his assistance in the determination of serum prolactin concentrations. Mrs. Marilyn Schurman and Mr. Jim Liesman for their competent assis- tance in the laboratory. iii Dr. C. A. Lassiter and the Institute of Nutrition for providing funds in the form of a research assistantship and NIH Traineeship. Especially grateful thanks are extended to my wife, Lu, and daughters, Johnna and Rebekah, for their patience, understanding, and unselfish- ness throughout this study. iv VITA John Edd Shirley was born January 26, 1943 in Monroe County, Kentucky. He attended Sand Lick Elementary School and Tompkinsville High School. He served 4 years in the United States Air Force. He received the Bachelor of Science Degree in Agriculture from Western Kentucky University in August 1968, and the Master of Science Degree in Agriculture in February 1970. His Master of Science Degree was done under the supervision of Dr. L. D. Brown. The title of his thesis was "Influence of Urea on the Fermentation Pattern and Nutri- tive Value of Corn Silage." He will receive a Doctor of Philosophy Degree from the Department of Dairy Science and the Institute of Nutrition in 1973. He will join the faculty of the Agriculture De-. partment at Eastern Kentucky University, Richmond, Kentucky in August 1973 as an assistant professor. TABLE OF CONTENTS Page LIST OF TABLES .......................... x LIST OF FIGURES ......................... xii LIST OF APPENDICES ........................ xiii INTRODUCTION ........................... 1 REVIEW OF LITERATURE ....................... 4 Introduction ......................... 4 Uptake of Plasma Triglycerides ................ 4 Mammary Gland ...................... 5 Adipose Tissue ...................... 7 Regulation of Plasma Triglyceride Uptake by Extrahepatic Tissues .......................... 8 Lipoprotein Lipase .................... 9 Glyceride Synthesis ................... 15 Plasma Lipoprotein Lipase .................. 19 Source .......................... 20' Clearance from the Circulatory System .......... 21 vi TABLE OF CONTENTS (cont.) Page Lipases Other than Lipoprotein Lipase in Postheparin Plasma ......................... 23 Contribution of Plasma Lipid to Milk Fat ........... 24 Effect of Stage of Lactation on the Amount and Composition of Milk Fat ........................ 26 METHODS AND MATERIALS ...................... 29 Studies on Adipose and Mammary Tissues ............ 29 Sample Collection Techniques ............... 29 Adipose tissue biopsy ................ 29 Mammary tissue biopsy ................ 29 Slaughter samples .................. 31 Tissue handling procedures .............. 31/ Preparation of tissue for enzyme assays ....... 32 Enzyme Assays ...................... 32 Experiment T1 .................... 32 Experiments T2 and P2 ................ 33 Experimental Procedures . . . . . . . . . . . . ..... 39 Experiment T]: Effect of induced lactation on enzymic activity in primiparous bovines ...... 39 Experiment T2: Enzymic activity during various lactational states in multiparous bovines ..... 40 Studies on Plasma Lipolytic Activity ............. 42 Plasma Sample Collection and Treatment .......... 42 Postheparin Plasma Samples ................ 43 Mammary Arterio-Venous Technique ............. 43 vii TABLE OF CONTENTS (cont.) Page Plasma Lipolytic Activity Assay ............. 44 Experimental Procedures ................. 45 Experiment P1: Plasma lipolytic activity vs milk fat production ................... 45 Experiment P2: Plasma lipolytic activity vs adipose and mammary tissue enzymic activity . . . . 50 Experiment P3: Effect of initiation of lactation on plasma lipolytic activity ............ 50 Experiment P4: Effect of time after milking on plasma lipolytic activity ............. 51 Other Methods ........................ 52 RESULTS AND DISCUSSION ...................... 53 Tissue Studies ........................ 53 Introduction ....................... 53 Experiment T1 ...................... 55 General ....................... 55 Protein and dry matter ................ 55 Lipoprotein lipase and glyceride synthesis ...... 55 Experiment T2 ...................... 58 Mammary gland .................... 58 Adipose tissue .................... 61 Plasma Studies ........................ 65 Plasma Lipolytic Activity vs Milk Fat Production ..... 65 Effect of Mammary Gland Emptying on Lipolytic Activity in Mammary Venous Plasma ................ 68 Relationship between Plasma Lipolytic Activity and Energy Status ..................... 71 viii TABLE OF CONTENTS (cont.) Page Effect of Induction of Lactation on Plasma Lipolytic Activity ........................ 76 Plasma Lipolytic Activity vs Adipose and Mammary LPL and GS Activity .................... 80 GENERAL DISCUSSION ........................ 87 SUMMARY ............................. 94 APPENDICES ............................ 98 BIBLIOGRAPHY ........................... 104 ix LIST OF TABLES Table Page 1. IN VITRO ASSAY SYSTEM FOR BOVINE ADIPOSE AND MAMMARY LIPOPROTEIN LIPASE .................... 35 2. EXPERIMENT T2 SAMPLING DATES ................ 41 3. IN VITRO ASSAY SYSTEM FOR PLASMA LIPOLYTIC ACTIVITY . . . . 44 4. LIPID METABOLISM IN PRIMIPAROUS BOVINE MAMMARY AND ADIPOSE TISSUE ...................... 56 5. MAMMARY LIPOPROTEIN LIPASE RESPONSE TO HEPARIN ....... 57 6. CORRELATION 0F PLASMA LIPOLYTIC ACTIVITIES WITH MILK AND MILK FAT PRODUCTION ................... 67 7. EFFECT OF MAMMARY GLAND EMPTYING ON LIPOLYTIC ACTIVITY IN MAMMARY VENOUS PLASMA ................. 69 8. PROLACTIN RESPONSE TO MILKING STIMULUS ........... 71 9. CORRELATION OF ENERGY STATUS WITH MILK FAT PRODUCTION AND PLASMA LIPOLYTIC ACTIVITIES ............. 75 10. PLASMA LIPOLYTIC ACTIVITY ACROSS PARTURITION IN PRIMIPAROUS HEIFERS ................... 77 11. PEAK POSTHEPARIN PLASMA LIPOLYTIC ACTIVITY ACROSS PARTURITION IN PRIMIPAROUS HEIFERS ............ 79 12. RELATIONSHIP BETWEEN PLASMA LIPOLYTIC ACTIVITY AND MAMMARY TISSUE ENZYME ACTIVITIES AT VARIOUS LACTATIONAL STATES .................... 81 LIST OF TABLES (cont.) Table Page 13. RELATIONSHIP BETWEEN PLASMA LIPOLYTIC ACTIVITY AND ADIPOSE TISSUE ENZYME ACTIVITIES AT VARIOUS LACTATIONAL STATES .................... 82 xi Figure 1. LIST OF FIGURES Lipoprotein lipase activity in homogenates of adipose tissue at different incubation times ........... Lipoprotein lipase activity in homogenates of mammary tissue at different incubation times ......... Plasma lipolytic activity at different incubation times . Postheparin plasma lipolytic activity at different incubation times ..................... Activities of mammary tissue lipoprotein lipase and glyceride synthesis during a complete lactational cycle . Activities of adipose tissue lipoprotein lipase and gly- ceride synthesis during a complete lactational cycle. . . Temporal response of plasma lipolytic activity to pro— lactin injection ................... Pre- and postheparin lipolytic activity in mammary venous plasma and mammary tissue lipoprotein lipase activity across parturition ..... . . ............. xii Page 37 37 46 48 62 72 LIST OF APPENDICES Appendix Page A. COMPOSITION OF SCINTILLATION FLUID ............ 98 B. TEMPORAL RESPONSE OF PLASMA LIPOLYTIC ACTIVITY TO INTRAVENOUS INJECTION 0F HEPARIN ............ 99 C. ADIPOSE AND MAMMARY LPL RESPONSE TO HEPARIN (EXPERIMENT T2) ..................... 100 D. INDIVIDUAL COW DATA FROM EXPERIMENT P] .......... 102 E. PLASMA LIPOLYTIC ACTIVITY AND ACTIVITIES 0F ADIPOSE AND MAMMARY TISSUE LPL AND GS (EXPERIMENT P2) ........ 103 xiii INTRODUCTION The non-lactating ruminant mammary gland depends primarily on the utilization of acetate for energy. With lactogenesis, there is a sharp increase in utilization of blood sugar, ketone bodies, and tri- glycerides. This increased utilization of nutrients by the mammary gland shifts the animal into negative energy balance with a resultant increase in fat mobilization from body stores. This situation con- tinues throughout early lactation, presumably because the animal is unable to consume sufficient energy to meet her needs for maintenance and milk production. The energy inputzoutput ratio becomes more favor- able as lactation advances and the animal begins to replete body energy stores. This synopsis serves to point out the continual competition for calories between adipose tissue and the mammary gland during lacta- tion. The mammary gland has the advantage during early lactation, a balance is achieved at midlactation, and adipose tissue is favored dur- ing late lactation. This relationship has been somewhat characterized in the literature in terms of variation in plasma lipid and milk fat at various stages of lactation. Little information is available on the enzymic changes behind this observed phenomenon. l Several metabolic alterations may contribute to this adipose- mammary relationship. However, this investigation is primarily directed toward the influence exerted by the enzymes responsible for uptake of plasma triglyceride fatty acid by adipose and mammary tissues. Funda- mentally, uptake involves extracellular hydrolysis of triglyceride to free fatty acids and glycerol, and subsequent reesterification within the cell. Lipoprotein lipase (LPL) is generally accepted to be respon- sible for the hydrolytic step and glyceride synthesis (either a- glycerol phosphate or 2-monoglyceride pathway or both) for reesterifi- cation within the cell. The relative influence exerted by adipose and mammary LPL and glyceride synthesis on the diversion of blood fat to the mammary gland has been investigated in nutritionally milk fat- depressed cows. These studies established that LPL and glyceride syn- thesis are involved in plasma glyceride fatty acid uptake by bovine adipose and mammary tissues. However, they provided little insight into the specific role each played with respect to regulation of milk fat production. Data from other species indicating that initiation of lacta- tion has profound effects on activities of both adipose and mammary LPL and glyceride synthesis suggest an alternative means of investi- gating their role with respect to milk fat production. The method suggested is the investigation of the effects of gestation, parturition, and subsequent lactation on adipose and mammary tissue LPL and gly- ceride synthesis activities in the bovine. Various workers have pointed out that LPL activity in tissue homogenates Or extraCts may not reflect the physiologiCal activity of the enzymes.. This conclusion is based on data suggesting that LPL exists in the cell in‘a state of reduced activity, is activated just prior to or during release from the cell and functions at the capil- lary membrane. Thus, a more accurate measure of phySiological LPL activity may be that activity in the circulatory system. The primary objectives of this study were to establish: 1) the importance of adipose and mammary triglyceride uptake ability relative to distribution of lipid between the two tissues; 2) the importance of LPL versus glyceride synthesis in regulation of trigly- ceride uptake; 3) the feasibility of using mammary venous plasma tri— glyceride lipase activity as an indicator of the mammary gland's ability to clear triglyceride fatty acids from the bloodstream. REVIEW OF LITERATURE Introduction The intent of this review is to bring together pertinent infor- mation on the mechanism employed by extrahepatic tissues to clear lipoprotein triglycerides from plasma and possible regulators of plasma triglyceride uptake by these tissues. Emphasis will be placed on tri- ‘glyceride Uptake by adipose tissue and the mammary gland. A consider— - ation of plasma lipoprotein lipase will be presented with emphasis on“ the origin of plasma lipoprotein lipase and the way it is cleared from the bloodstream. The contribution of plasma lipid to milk fat and effects of stage of lactation on milk fat composition will receive brief consideration. Uptake of Plasma Triglycerides Long chain fatty acids serve as a readily utilizable form of energy for peripheral tissues. They are derived from the diet or from de novo synthesis in liver and adipose tissue and transported in the blood stream as triglycerides of chylomicra and low density lipoproteins (Scow et al., 1972; Griel and McCarthy, 1969). These triglycerides are cleared from the blood stream by the liver and by extrahepatic tissues (Shapiro, 1965). The mechanism by which extrahepatic tissues, such as adipose and mammary, clear such large particles as chylomicra and low density lipoproteins from the blood stream has generated considerable interest in recent years. Adipose and mamnary tissue capillaries have a continuous luminal surface which cannot accommodate the passage of intact chylomicra and other lipid particles (Scow, 1970). This section explores the mechanism used by adipose tissue and the mammary gland to clear triglycerides from the blood stream. Mammary Gland The majority of investigations indicate that hydrolysis of triglycerides to free fatty acids and glycerol occurs prior to uptake by the mammocyte. Results obtained by infusing doubly labeled chylomicra into guinea pig (McBride and Korn, 1964b), goat (West et al., 1967a, 1967b), and c0w (Bishop et al., 1969) are consistent with this view. Collectively, they found a decrease in the labeled glycerolzfatty acid ratio in milk fat relative to infused chylomicron triglycerides and the appearance of labeled non-esterified fatty acids in mammary venous blood. To determine where hydrolysis occurs during uptake, Mendelson and Scow (1972) perfused the inguinal abdominal mam- mary glands of lactating rats in situ with doubly labeled chylomicron- 14C-palmitate). The temporal relation- triglyceride (3H-glycerol and ship between infusion and appearance of 14C-palmitate in the perfusate leaving the gland was observed. They were able to conclude that hy- drolysis occurs at or within the capillary membrane. Further evidence for hydrolysis at the capillary membrane was provided by electron micrographs of lactating mouse mammary tissue (Schoefl and French, 1968). These micrographs showed adhesion and engulfment of chylomicra by the inner membrane of endothelial cells of mammary capillaries with no sign of lipid globules outside the membrane. Lipoprotein lipase has been implicated as the enzyme respon- sible for hydrolysis of chylomicra and low density lipoprotein trigly- cerides prior to uptake. Several workers observed a close correlation between mammary lipoprotein lipase activity and uptake of plasma tri- glycerides (McBride and Korn, 1963; McBride and Korn, 1964a; Robinson, 1963a; Hamosh et al., 1970). The non-lactating mammary gland of the rat (Hamosh et al., 1970), guinea pig (McBride and Korn, 1963 and 1964a; Robinson, 1963a), rabbit (Falconer and Fiddler, 1970), and cow (Askew et al., 1970) contain very low levels of lipoprotein lipase activity. Initiation of lactation and subsequent need for milk fat precursors is accompanied by a large increase in lipoprdtein lipase activity. Conversely, nonsuckling results in a decrease in mammary tissue lipoprotein lipase activity in guinea pigs (McBride and Korn, 1963) and rats (Hamosh et al., 1970) and an increased plasma trigly- ceride concentration. Adipose Tissue Clearance of triglyceride fatty acids from blood by adipose tissue requires prior hydrolysis to free fatty acids and glycerol (McBride and Korn, 1964b; Robinson, 1963b; Scow et al., 1972). The extent of lipoprotein lipase involvement in blood triglyceride clear- ance is indicated by adipose tissue uptake of some 30 per cent of the fatty acids entering the blood of animals in positive caloric balance and in those absorbing fatty meals (Bragdon and Gordon, 1958). This uptake occurs directly from triglyceride fatty acids of chylomicra in blood (Nestel et al., 1962a, 1962b). On the other hand, in fasted animals triglyceride fatty acids in blood are not take up by adipose tissue (Bragdon and Gordon, 1958) and lipoprotein lipase activity in adipose tissue decreases (Hollenberg, 1959; Robinson, 1960; Pav and Wenkeova, 1960). Uptake of triglyceride fatty acids by adipose tissue varies with the tissue level of lipoprotein lipase activity (Bezman et al., 1962). Adipocytes and vascular-stromal cells of adipose tissue both contain lipoprotein lipase (Rodbell and Scow, 1965; Cunningham and Robinson, 1969). Release of lipoprotein lipase into the blood stream can be accomplished by heparin infusion (Rodbell and Scow, 1965). Per- fusion of adipose tissue with heparin results in a rapid release of lipoprotein lipase into the perfusate and a subsequent decrease in the rate of hydrolysis of chylomicra triglycerides (Scow et al., 1972). Since heparin presumably causes release of the lipoprotein lipase located within or near the capillary wall (Ho et al., 1967; Rodbell and Scow, 1965) it appears that the site of hydrolysis is at the capil-‘ lary membrane. Blanchette-Mackie and Scow (1971), using the electron microscope and cytochemical techniques, provided convincing evidence for hydrolysis at the capillary membrane. Their findings indicate that triglycerides are hydrolyzed by lipoprotein lipase in vacuoles and microvesicles of the capillary endothelium and in the subendothelial space of adipose tissue. Regulation of Plasma Triglyceride Fatty Acid Uptake by Extrahepatic Tissues Long chain fatty acids, transported through the blood stream as chylomicra and B-lipoproteins, are precursors for approximately : 50 per cent of milk fat triglycerides. During lactation, the mammary gland competes with other extrahepatic tissues for these triglycerides. Predominate among the mammary gland competitors is adipose tissue. The mechanism by which extrahepatic tissues remove chylomicra and low den- sity lipoprotein triglycerides from circulation involves lipoprotein lipase and glyceride synthesis. Either one of these enzymic systems could directly influence uptake and consequently the final destination of circulating triglycerides. It is recognized that other factors could influence final destination of blood triglycerides but this re- view will be confined to possible regulation of uptake at the tissue level. Lipoprotein lipase and glyceride synthesis as possible regula- tory agents will be discussed separately. LipOprotein Lipase LipOprotein lipase (LPL) is a triglyceride lipase with prefer- ential activity against triglycerides of lipoprotein origin. It is active against artificial triglyceride (triolein, etc.) emulsions if the emulsion is converted to an active lipoprotein substrate by addi- tion of serum, lipoprotein fractions, or specific serum peptides from lipoproteins (Korn, 1955b; Fielding et al., 1970; Havel et al., 1970; LaRosa et al., 1970; Whayne and Felts, 1970; Miller and Smith, 1973). Contrary to early reports (Korn, 1955a), the major products of hy- drolysis by LPL appear to be free fatty acids and 2-monoglyceride 10 (Nilsson-Ehle et al., 1971; Fielding, 1972). Although miner activity against monoglyceride substrates was observed, these contrasting re- sults are apparently due to the state of purification of the enzyme. The assay of LPL activity in tissue homogenates and extracts, and in plasma requires: 1) a serum activated triglyceride substrate; 2) a pH optima between 8.0 and 8.6, depending on the enzyme prepara- tion and the nature of the substrate (Fielding, 1972; Korn, 1955a,b; Whayne and Felts, 1972); 3) a free fatty acid acceptor such as albumin (Gordon et al., 1953; Korn, 1955a); and 4) the presence of divalent cations, in particular calcium at concentrations found in serum.' Addition of low concentrations of heparin to the assay medium enhances LPL activity while high concentrations inhibit activity (Korn, 1955a). A specific requirement for ammonium ions in the assay of LPL activity has been reported (Korn, 1955a) and refuted (Whayne and Felts, 1971). It is generally accepted that LPL functions in the uptake of plasma triglyceride fatty acids by extrahepatic tissues. Prominent among these being muscle, adipose, mammary, and cardiovascular (Scow, 1970; Korn, 1959; Robinson, 1963a). LPL appears to exist in the cell in a state of reduced activity (Cunningham and Robinson, 1969; Robin- son and Wing, 1970) and is activated just prior to or during release from the cell (Stewart and Schotz, 1971). The functional site of 11 action appears to be at the capillary membrane (West et al., 1967a,b; Robinson and Wing, 1970; Scow et al., 1972; Schoefl and French, 1968). The importance of LPL in the removal of triglycerides from the blood is illustrated by Familial Type I hyperlipoproteinemia in which LPL is absent or present in residual levels (Fredrickson and Levy, 1972). Further, the hypertriglyceridemia associated with diabetes mellitus may be due partly to a decrease in adipose tissue LPL activ- ity (Kessler, 1963). Hypertriglyceridemic conditions associated with oral contraceptives have been characterized partially by decreased postheparin lipolytic activity in plasma. Postheparin lipolytic ac- tivity is partially due to LPL released from extrahepatic tissues (Korn, 1955; Payza et al., 1967). LPL deficiency has also been im- plicated in the lipemia associated with late gestation and parturi- tion (Otway and Robinson, 1968). The extent of LPL involvement in blood triglyceride clearance is indicated by adipose tissue uptake of some 30 per cent of the fatty acids entering the blood in animals in positive caloric balance and in those absorbing fatty meals (Bragdon and Gordon, 1958). This uptake occurs directly from triglyceride fatty acids of chylomicra in blood (Nestel et al., 1962a,b). On the other hand, in fasted animals tri- glyceride fatty acids in blood are not taken up by adipose tissue (Bragdon and Gordon, 1958) and LPL activity in adipose tissue 12 decreases (Hollenberg, 1959; Robinson, 1960; Pav and Wenkeova, 1960). Uptake of triglyceride fatty acids by adipose tissue varies with the tissue level of LPL activity (Bezman et al., 1962). LPL activity (Hamosh et al., 1970; McBride and Korn, 1963; Robinson, 1963a) and blood glyceride fatty acid uptake (Barry et al., 1963; McBride and Korn, 1964a) are both increased in mammary tissue with initiation of lactation. Although processes other than hydrolysis by LPL are in- volved in glyceride fatty acid uptake (Shapiro, 1965) by adipose and, presumably, mammary tissue, it is clear that LPL plays an important role in uptake. The parallel relationship between LPL activity and glyceride fatty acid uptake by adipose and mammary tissues suggest that the enzyme serves in a regulatory or directive capacity. Such a role is supported by the fact that lipoprotein lipase activity changes with physiological state of the animal. The direction of change is depen- dant on the physiological state and the tissue in question. Thus, during fasting and lactation when the need for energy mobilization is high, adipose LPL activity is decreased (Hollenberg, 1959; Robin- son, 1960; Hamosh et al., 1970). On the other hand, fasting animals have elevated LPL activity in heart and skeletal muscle (Hollenberg, 1960; Borensztajn and Robinson, 1970; Borensztajn et al., 1970) and initiation of lactation results in highly elevated LPL activity in 13 mammary tissue (Hamosh et al., 1970; McBride and Korn, 1963; Robinson, 1963a; Askew et al., 1970). Conversely when the body returns to a positive energy balance adipose LPL activity increases. Much of the work on regulation of LPL has involved adipose tissue as the source of enzyme and has centered around the effects of fasting and refeeding. Robinson and Wing (1970) pointed out that insulin is an effective stimulator of LPL activity when pieces of adi- pose tissue from fasted but not from fed rats are incubated in vitro. This effect was blocked by adrenaline, noradrenaline, and adrenocorti- cotrophic hormone. Glucagon and thyroid stimulating hormones have also been shown to block the stimulatory effects of insulin on LPL activity in adipose tissue from fasted rats (Nestel and Austin, 1969). That insulin stimulates formation rather than release of adipose LPL has been confirmed with the dispersed cell (adipocytes) technique (Robinson and Wing, 1970). Investigations of mammary LPL have centered around the in- crease in activity noted at initiation of lactation (Hamosh et al., 1970; McBride and Korn, 1963; Robinson, 1963a). The many hormonal changes taking place at this time (Convey, 1973) suggest hormonal regulation of mammary LPL. Pursuing this possibility, Falconer and Fiddler (1970) found that prolactin administrated intraductally 14 induced\tPL synthesis and subsequent increase in aCtivity in the pseudo- pregnant rabbit mammary gland. Several indirect observations of steroid hormone effects on LPL have been reported. Hazzard et a1. (1969) and Adams et a1. (1970) found postheparin lipolytic activity to be depressed in normal women taking mixed oral contraceptives. The estrogen component appeared to have the depressing effect (Hazzard et al., 1969). Patients receiving parenteral estradiol therapy have also been reported to have depressed postheparin lipolytic activity (Fabian et al., 1968). In contrast, synthetic ster- oids with progestational activity such as norethindione increase plasma postheparin lipolytic activity and decrease plasma triglycerides (Glueck et al., 1969; Glueck et al., 1972). Anabolic-androgenic compounds (oxandrolane) increase postheparin lipolytic activity and decrease plasma triglycerides (Glueck, 1971). LPL exhibits certain characteristics indicative of a regulatory enzyme: 1) it is located at the first step in uptake of blood trigly- ceride fatty acids by extrahepatic tissues; 2) it is responsive to normal, abnormal, and induced physiological changes; 3) its activity is correlated with uptake of blood triglyceride fatty acids by extra- hepatic tissues; 4) it is responsive to hormonal influences; and 5) different tissue LPL's respond differently to various stimuli 15 providing a means of influencing final destinations of blood trigly- ceride fatty acids depending on need. Glyceride Synthesis A very important aspect of adipose and mammary physiology is their ability to synthesize triglycerides from free fatty acids and glycerol. This provides adipose tissue with the ability to store not only newly synthesized fatty acids but those cleared from circulation. Glyceride synthesizing capability provides the mammary gland with the ability to utilize both de novo synthesized fatty acids and those pre— sented to the tissue via the blood stream. Fatty acid esterification in bovine adipose and mammary tissues has recently been characterized and reviewed (Storry, 1970; Benson and Emery, 1971; Askew et al., 1971; Bickerstaffe, 1971) and will receive only brief mention here. The predominant pathway for glyceride synthesis in mammary tissue is the glycerol phosphate pathway (Storry, 1970; Bickerstaffe, 1971). Glyceride synthesis in the bovine (Kinsella, 1968; Askew et al., 1971) mammary gland occurs in the particulate fraction of the cell and is dependant upon adenosine triphosphate, coenzyme A, glycerol-3- phosphate, and magnesium. Although these results are consistent with esterification via the glycerol phosphate pathway, it has been 16 suggested that the monoglyceride pathway may also be operational in mammary tissue (Bickerstaffe et al., 1970; Dimick et al., 1970). Fatty acid esterification in adipose tissue occurs predomi- nantly, if not exclusively, via the glycerol phosphate pathway (Sha- piro, 1965). Glyceride synthesis in homogenates of bovine adipose tissue requires glycerol-3-phosphate, adenosine triphosphate, coen- zyme A, and magnesium chloride as cofactors (Benson and Emery, 1971). Similar requirements have been reported for fatty acid esterification by rat adipose tissue (Steinberg et al., 1961; Angel and Roncari, 1967). Regulation of triglyceride fatty acid uptake by adipose and mammary tissues is often considered the domain of LPL. However, gly- ceride synthesis may function synchronously with LPL in the assimila— tion of circulating triglyceride fatty acids (Dole, 1961) and could regulate uptake via end product inhibition of LPL. In other words, a decrease in esterification rate could result in a local buildup of nonesterified fatty acids. There is evidence that unbound free fatty acids inhibit the lipolytic activity of LPL (Gordon et al., 1953). Nikkila and Pykalisto (1968) have suggested that the free fatty acid concentration in the tissue may be the actual effector of LPL. This observation was based on the ability of nicotinic acid to increase LPL activity via a decrease in fat mobilization. Wing and Robinson 17 (1968) have investigated the relationship between tissue free fatty acid concentration and LPL activity in a series of experiments using epididymal fat pads from rats. Their results include: 1) a rapid decrease in LPL activity during starvation accompanied by a rise in tissue free fatty acid concentration, 2) dibutyryl cyclic AMP (lipo— lytic agent) inhibition of the normal increase in LPL activity when adipose tissue from fasted rats was incubated in a complete medium with inhibition being greater at low glucose concentration, 3) caf— feine plus dibutyryl cyclic AMP inhibition of the normal increase in LPL activity irrespective of glucose concentration up to 2.4 mg/ml. (A large increase in tissue and medium free fatty acids were also observed.) 4) caffeine (5mM) inhibition of the normal increase in LPL activity without any effect on concentration of free fatty acids in the tissue or medium. These results with the possible exception of those obtained with 5 mM caffeine are all consistent with the hypothesis of end product inhibition of LPL. The effect of glucose concentration on the degree of inhibition mediated by dibutyryl cyclic AMP is of particular interest since esterification in adipose tissue proceeds predominantely through the d-glycerol phosphate pathway. Glucose availability at the cellular level could increase the a-glycerol phosphate concentration which in turn could stimulate esterification, 18 thus pulling the reaction in favor of increased LPL activity. Whether or not this could create an observable effect on LPL activity within the three hour incubation period used is a matter of conjecture at this point. However, glucose, fructose, and galactose have been observed to decrease free fatty acid release from adipose tissue in vitro with- out influencing the release of glycerol (Leboeufet al., 1959; Buckle et al., 1961; Vaughan, 1962; Gorin and Shafrir, 1963). These results were attributed to an increase in a-glycerol phosphate and possibly an increase in energy, both of which would tend to increase esterifi— cation, thus a recycling of endogenously released free fatty acids. There is evidence indicating that conditions conducive to high LPL activity are also conducive for high rates of fatty acid esteri- fication. Ball and Jungas (1963) and Angel and Roncari (1967) re- ported that fasting decreases and refeeding increases glyceride syn- thesis (GS) activity in adipose tissue. Similar responses are ob- served in LPL activity (Hollenberg, 1959; Robinson, 1960). Addition of glucose to the incubation medium enhances fatty acid esterification by adipose tissue (Steinberg and Vaughan, 1965). Further enhancement of GS is achieved if insulin is added in the presence of glucose. Similar observations have been reported with respect to LPL (Hollen- berg, 1959; Salaman and Robinson, 1966; Austin and Nestel, 1968). The feeding of restricted roughage-high grain diets stimulated the 19 activities of both GS and LPL in bovine adipose tissue (Benson et al., 1972). These diets tend to elevate blood glucose (Jorgensen et al., 1965; Storry and Rook, 1966). Thus far, the evidence indicates that in adipose tissue and, presumably, mammary gland the uptake and reesterification of blood triglyceride fatty acids is closely associated with carbohydrate metabolism, that LPL and GS respond in a similar manner to physiolog— ical changes, and that either of these two systems could regulate clearance of chylomicra and low density lipoprotein triglycerides from the blood. Plasma Lipoprotein Lipase In 1943, Hahn (1943) reported that injection of heparin intra- venously results in rapid clearing of alimentary lipemia. Postheparin plasma was later found to contain a factor which cleared lipemia in vitro (Anderson and Fawcett, 1950). Shortly thereafter, Korn (1955a) established that the clearing factor in postheparin plasma was the enzyme lipoprotein lipase (LPL). Korn (1955a) further reported that LPL catalyzes the hydrolysis of the triglyceride moiety of chylomicra and low density lipoproteins to free fatty acids and glycerol. 20 These early reports provided impetus for a multiplicity of studies on plasma postheparin lipolytic activity. Normally plasma contains very small amounts of LPL (Engelberg, 1956; Korn et al., 1961). However, lipolytic activity rises markedly within minutes after intravenous injection of heparin (Korn, 1961; Porte and Wil- liams, 1965) and then disappears exponentially. The rate of increase and level of enzyme activity achieved appeares to be dependent on the amount of heparin injected. Maximum plasma enzyme activity was ob— served within 10 minutes following intravenous injection of 10,000 International Units (IU) of sodium heparin into human subjects (Field- ing, 1970a). LaRosa et a1. (1971) found maximum activity between 30 and 240 minutes after injection of 5,000 IU of Na Heparin. Source LPL was first observed in plasma following intravenous injec- tion of heparin. Subsequent studies revealed the presence of an enzyme . with similar properties in various extrahepatic tissues including; heart (Korn and Quigley, 1955), mammary gland (McBride and Korn, 1963; Robinson, 1963a), adipose (Korn and Quigley, 1955; 1957), lung (Brady and Higgins, 1967), and skeletal muscle (Olivecrona and Belfrage, 1965; Wallach, 1968). The brain and pancreas apparently lack LPL activity (Swank and Levy, 1952; Miller, 1969). 21 The similarity between plasma clearing factor lipase and tissue LPL led to the hypothesis that the source of the plasma enzyme was the various extrahepatic tissues. This hypothesis was substantiated by em- ploying in vitro and in situ organ perfusion techniques using heparin in the perfusate. Heart, skeletal muscle, adipose tissue, and the mammary gland have all been shown to contribute LPL to the circulation (Lequire et al., 1963; Robinson et al., 1963; Robinson and Jennings, 1965; Rodbell and Scow, 1965; Enser et al., 1967; Ho et al., 1967; Nestel, 1970). Clearance from the Circulatory System Liver (Spitzer and Spitzer, 1956; Connor and Eckstein, 1959) and kidney (Constantinides et al., 1959) have been implicated as being the primary sites of rembval of LPL from circulation. Clearance of plasma postheparin lipolytic activity is markedly slowed by hepatectomy (Yoshitashi et al., 1963), fatty livers (dePury and Collins, 1972), carbon tetrachloride cirrhotic livers, and nephrectomy (Constantinides et al., 1959). Homogenates of liver are inhibitory to plasma post- heparin LPL activity (Yoshitashi et al., 1963). Acetone powder prepar- ations of rat liver have been shown to inhibit LPL activity from heart muscle (Mayes and Felts, 1968). LPL activity has also been reported to be inhibited by kidney extracts (Klein et al., 1958). 22 These data indicate that the liver and kidney are important in clearance of LPL from circulation. Since extracts of these tissues in- hibit LPL activity from other sources, it would appear that this is accomplished by a secreted factor. A well designed investigation by Naito and Felts (1970) further established the role of the liver in plasma LPL clearance and provided insight into the mechanism employed. They proposed a two—step inactivation system for LPL by liver. The first step involves the dissociation of a heparin—apoenzyme complex followed by destruction of heparin. The second step involves the re- moval of the apoenzyme of LPL. Evidence for the second step was weak but the first step was indicated by several points. When heparin was added to the perfusate pridr to passage through the liver it partially blocked clearance suggesting that the heparin removal mechanism became saturated. Heparin added to the liver effluent stimulated LPL activity more than-normal but not to preperfusion levels. This indicates he- parin removal from the enzyme during passage through the liver. Heparinase could well be the agent involved in this step since its presence in liver has long been known (Jacques, 1940). Heparinase could also be the inhibitory factor associated with liver and kidney homogenates and extracts as mentioned earlier. Pertinent to a discussion of the ability of an organ to clear a substance from circulation is its effectiveness. The effectiveness 23 of the liver to clear LPL has been studied by Whayne et a1. (1969) and Naito and Felts (1970). Whayne et a1. (1969) reported the extraction ratios across the livers of two dogs were .68 and .42 (extraction ratio being the inputzoutput ratio). Similar values (.70) were obtained by perfusion studies with rat livers (Naito and Felts, 1970). Lipases Other than Lipoprotein Lipase in Postheparin Plasma The multiplicity of hydrOlytic activities in postheparin plasma (Shore and Shore, 1961; Fielding, 1970a; Greten et al., 1970; Vogel and Bierman, 1970; Nilsson-Ehle et al., 1971; Smith, 1972; Jansen and Huls- mann, 1973) suggests the presence of more than one enzyme. Differential studies of postheparin plasma lipolytic activity have indicated the presence of not only LPL activity but activities of: l) long-chain acyl-CoA hydrolase (Jansen and Hulsmann, 1973), 2) a monoglyceride hydrolase (Shore and Shore, 1961; Greten et al., 1969; Nilsson—Ehle and Belfrage, 1972), 3) a diglyceride lipase (Greten et al., 1970), and 4) a hepatic triglyceride lipase (LaRosa et al., 1972). 24 Contribution of Plasma Lipid to Milk Fat A positive relationship exists during the lactation cycle be- tween the level of blood lipid and the amount of milk produced. Blood lipids rise to a high level following parturition and remain high dur- ing the period of greatest milk production. The level of blood lipids gradually declines as milk production diminishes, reaching a low level as the dry period approaches. The effect of lactation on the level of blood lipids was found to be independent of effects of season and die- tary fat. These early observations by Maynard et a1. (1931) initiated a renewed interest in the contribution of plasma lipids to milk fat in the bovine. Earlier studies by Meigs et a1. (1919) had suggested that blood phospholipids were the source of milk fat. They (Meigs et al., 1919) reported that marrmary venous blood contained less phospholipid and more inorganic phosphorus than was present in the general circula- tion. In some cases they were able to account for the total milk fat secreted by the decrease in phospholipids in blood as it passed through the mammary gland. McCay and Maynard (1935) reinvestigated the uptake of phospholipid by the mammary gland and found no significant differ- ence between the phospholipid content of jugular and arterial blood and that.of the mammary venous blood. This observation was confirmed the f'CJllowing year by Graham et a1. (1936). Further investigations of 25 arterio-venous levels of blood lipids across the mammary gland caused Shaw and Petersen (l940a,b) and Voris et a1. (1940) to conclude that blood neutral fat and glycerides are absorbed but probably no other fraction with the possible exception of cholesterol esters. Shaw and Petersen (1938) concluded that the mammary gland removed enough fat from blood to account for the milk fat plus an additional supply to be oxidized for energy. However, followup studies (Shaw and Petersen, 1940a,b; Shaw and Knodt, 1941) with improved techniques failed to con- firm mammary gland removal of fat from the blood in excess of that ex- creted in the milk. More recently, Riis (Ph.D. thesis as quoted by Storry, 1970) reported a positive correlation between the concentration of plasma phospholipids and milk fat production. Thus, resubmitting the sugges- tion that plasma phospholipids were contributors to milk fat. However, subsequent studies (Hartmann and Lascelles, 1964; Annison et al., 1967; West at al., 1967a; Varman and Schultz, 1968a; Bishop et al., 1969) have shown that plasma phospholipids and sterol esters are not utilized by the mammary gland. Techniques, including the feeding of radioactive triglycerides to cows (Glascock et al., 1966; Bishop et al., 1969), arterio-venous difference studies in goats (Barry et al., 1963) and cows (Emery et al., 1965), and perfusion of isolated goat mammary gland (Lascelles et al., 1964; Linzell et al., 1967) have clearly demonstrated the importance 26 of chylomicra and low density lipoprotein triglycerides and nonesteri; fied fatty acids of plasma as milk fat precursors. Further credence is added to these reports by the fact that Robinson et a1. (1963) found no significant arterio-venous difference across the non—lactating mammary gland in plasma triglyceride fatty acids; while during lactation large arterio-venous differences were found in the combined triglycerides of chylomicra and low density lipoportein (d < 1.019) in plasma. From a quantitative standpoint, the triglyceride fraction of chylomicra and low density lipoporteins have been reported to contri- bute 50 to 60 per cent of the milk fat triglycerides in the goat (Linzell, 1968). In agreement with this, Bickerstaffe (1971) found that the respective contributions of chylomicra and low density lipo— protein triglycerides to milk fat were 14.7 and 35.7 per cent. He (Bickerstaffe, 1971) also reported that additional contributions of triglycerides were made by high density (9.8%) and very high density (15.5%) lipoportein fractions. Effect of Stage of Lactation on the Amount and Composition of Milk Fat Total daily milk production exhibits a characteristic increase for three to six weeks postpartum, followed by a gradual decline until 27 near the end of lactation (Brody et al., 1923; Turner et al., 1923; Drakeley and White, 1928). Fat content (per cent) varies inversely but not necessarily in direct proportion to milk production (Brown et al., 1962; Rook and Campling, 1965). The most marked changes in milk fat occur during the early postpartum period. In general, the proportion of C6_14 acids increase while the proportion of C acids decrease with time postpartum. 18 Little or no change occurs in the porportion of C acids (Boatman 16 et al., 1965; Stull and Brown, 1965). More specific information on proportional changes of individual fatty acids in milk fat during lactation has been reported by Stull et a1. (1966) using linear, quad- ratic, and cubic equations. The percentage of 6:0, 8:0, and 16:1 in milk fat during lactation was best described by a linear equation with a positive slope, and 16:0 by one with a negative slope. The percen- tage of 10:0 and 18:2 increased to mid-lactation then leveled off while 18:0 and 18:1 exhibited a negative initial slope with a slight upturn during the last 15 to 20 weeks of lactation. The percentage of 10:1, 12:1, 14:0, 14:1, 15:0, and 18:3 could all be defined by a cubic equa- tion; however, 10:1, 12:1, and 14:1 initially declined while the others increased. In terms of total yield, the C acids increase during 6-14 the first six weeks of lactation after which there is a steady decline. 28 The C acids appear to decline at a decreasing rate throughout 16-18 lactation (Storry, 1970). These data reflect two major events: 1) a gradual increase in de novo synthesis of milk fatty acids as indicated by the increase in C6-l4 acids during early lactation and 2) a decrease in fat mobil- ization from body stores with advancing lactation as evidenced by the decline in C acids. These conclusions are indicated since only 18 shorter chain acids (C4_]0) are derived entirely by mammary lipo- genesis while longer-chain acids (C18) of milk fat are derived en- tirely from plasma. The intermediate chain acids (C12-16) would be expected to fluctuate since they are derived from both sources (Garton, 1963; Barry, 1964; Linzell, 1968). The conclusion that degree of fat mobilization varies with stage of lactation and, thus, accounts for observed changes in propor- tions of long-chain acids in milk fat must be tempered with the ef- fects of diet (Van Soest, 1963) and season (Storry, 1970) on milk fat composition. METHODS AND MATERIALS Studies on Adipose and Mammary Tissues Sample Collection Techniques Adipose tissue biopsy: Subcutaneous adipose tissue samples were collected aseptically from an area posterior to the scapula near the dorsal midline. The area was clipped and scrubbed with an iodine solution (Betadine, Purdue Frederick Co., Norwalk, Connecticut). Ten mi of a 2.5% procaine (Procaine Hydrochloride Solution, Bio-ceutic Laboratories, St. Joseph, Missouri) solution was injected intrader- mally to anesthetize a circular area approximately 15 cm in diameter. After final preparation, the area was draped and a scalpel used to make a transverse 8 cm incision in the skin and subcutaneous tissue within the anesthetized area. Approximately 5 g of subcutaneous fat was removed. The skin was closed with interrupted 0.6 mm Vetafil (Vetafil Bengen, Haver-Lockhart, Kansas City, Missouri) sutures and sprayed with furazolidone (Topazone, Eaton Laboratories, Norwich, New York). Mammary tissue biopsy: Mammary tissue samples were collected aseptically at intervals not closer than one week. Milk from all 29 30 quarters was cultured and all quarters were free of pathogenic organ- isms prior to collecting the tissue. The cow was milked and the sur- gical field prepared as described for collecting samples of adipose tissue; however, the mammary gland was not anesthetized. The cow was placed on a hydraulic operating table in lateral recumbency. After final preparation, the area of the mammary gland was draped. A scalpel was used to make a 5 cm incision on the lateral surface of the mammary gland through the skin, subcutaneous tissue, and lateral suspensory ligament. A curved scissors was used to remove 5 g of mammary tissue. Hemostasis was established by ligating small blood vessels with 0 chromic_catgut (Ethicon, Somerville, New Jersey). An absorbable gela- tin sponge (Gelfoam, Upjohn Company, Kalamazoo, Michigan) was placed in the site of the excised tissue. Special care was taken in closing the lateral suspensory ligament with interrupted sutures of 0 chromic catgut (Ethicon, Somerville, New Jersey) to prevent subcutaneous hematomas. The skin incision was closed with 0.3 mm Vetafil (Vetafil Bengen, Haver-Lockhart, Kansas City, Missouri), sprayed with fura- zolidone (Topazone, Eaton Laboratories, Norwich, New York), and the cow was removed from the operating table. For three consecutive days postsurgery, prophylactic treat- ment consisted of procaine penicillin G (Procaine Penicillin G., W. A. Butler Co., Columbus, Ohio) (10,000 units/kg daily) injected 31 intramuscularly and an intramammary infusion of 15 m1 of Procaine Penicillin G-Furaltadone in oil (Altapen, Eaton Laboratories, Norwich, New York) into the operated quarter following the evening milking. Skin sutures were removed in seven days. Slagghter samples: Samples of adipose and mammary tissues were obtained in some cases following slaughter. In these instances, the animal(s) would betransported to the local slaughter house (Van Alstine) between 5:30 and 6:00 A.M. on the day of slaughter. Lactating cows were routinely milked prior to transport. The animals were rou- tinely sacrificed between 6:00 and 7:00 A.M. and representative samples of shoulder subcutaneous adipose and mammary tissues were obtained within 30 min after sacrifice. Tissue handling procedures: Samples of adipose tissue and tissue from mammary glands of non-lactating cows were rinsed in ice cold .15 M KCl to remove surface blood, blotted on paper towels, and placed in plastic bags in an ice bath for transport to the laboratory. All samples were frozen (within 1 hr after collection) at -40°C prior to measurement of lipoprotein lipase and glyceride synthesizing ac- tivities. Freezing at -40°C for up to six months has no significant effect on LPL activity (Askew et al., 1970). Mammary tissue samples from lactating cows were treated as previously described except they were initially rinsed in ice cold .15 M KCl containing 10 IU of 32 oxytocin (Syntocin, Sandoz pharmaceuticals, Hanover, New Jersey) to remove residual milk. Rinsing tissues from lactating cows with oxy- tocin decreased variation without significantly affecting LPL activity relative to KCl rinse only (4.2 i .3 vs 3.4 i .6 pmoles fatty acids released hr.1 mg protein-1, N = 5). Preparation of tissue for enzyme assays: Frozen mammary or adipose tissue was homogenized in .15 M KCl (pH 8.6) with a Polytron (Brinkman Instruments Inc., Des Plaines, Illinois) for l min and then centrifuged 10 min at 900 x g at 4°C. The supernatant was strained through cheese cloth and the filtrate used in the assays. Enzyme Assays Experiment 11: Lipoprotein lipase was assayed as outlined by Askew et a1. (1970) except fatty acids were measured colorimetrically (Mackenzie et al., 1967). Glyceride synthesis was measured by incor- 14 poration of C-labeled palmitate and oleate into glycerides as de- scribed by Benson and Emery (1971) with two modifications. An ATP generating system was added and the substrate concentration changed. Cofactor concentrations were as follows: ATP, 5 mM; DTT, 2mM; COA, .25 mM; MgCl 2.25 mM; KPO =, .05 mM; PEP, 5 mM; NaF, 15.5 mM; 2, a—GP, 20 mM; bovine serum albumin, 2.5 mg/mt, 12c-paimitic acid, 33 14C-palmitic acid, and 14C-oleic acid were added to give a final con- centration of .1 umoles/mt of each. Experiments T2 and P2: Glyceride synthesis was measured by incorporation of 14C-labeled palmitate and oleate as in Experiment T]. Lipoprotein lipase activity in tissue samples taken in these experiments was determined by the method of Schotz et a1. (1970) as modified herein. Blood obtained from the jugular vein of a lactating cow was allowed to clot for 2 hr at room temperature then refrigerated at 4°C overnight. Serum was separated by centrifugation and stored at 4°C until used (within 10 days). A mixture of 80 pCi of 3H—glyceryl trioleate and .133 g of unlabeled triolein was pipetted into plastic scintillation vials and dried in a sand bath under a stream of nitrogen to remove residual benzene. A mixture of .9 mt of 1% triton X-lOO plus 8.1 mi of buffer (pH 8.6) containing 8% bovine serum albumin, .24 M tris-HCl, and .15 M NaCl was added to the triolein. The mixture was placed in an ice bath and sonicated l min using a microtip on heat system-Ultrasonics model W 1850 operating at maximum power output (75 watts). The sonication step was repeated three times allowing one min for premix and sonic probe to cool between sonications. The resultant mixture was sufficient for the assay of 15 samples. When more than 15 samples were assayed, multiple batches of the substrate premix were prepared and the batthes mixed by sonication before use. 34 Two 10 pt aliquots of the substrate premix were transferred to scin- tillation vials to which .5 mt of an extracted water blank was added along with 10 mi of scintillation fluid (Appendix A). This served as a standard for computation of specific activity of substrate. The incubation mixture consisted of .6 mi of substrate premix, variable amounts of homogenate (.0-.2 mi), and .15 mt lactating serum. The mixture was made up to a total volume of 1.0 mt with .025 N NH4OH (pH 8.6). Serum blanks containing .15 mt of 3% bovine serum albumin (BSA Fraction V, Sigma Chemical Co., St. Louis, M0.) in .15 M NaCl (pH 7.4) instead of serum were routinely included in duplicate. Tissue samples were routinely assayed with and without heparin (.05 IU per assay); however, values obtained in absence of heparin are reported due to the large variation in heparin effect between tissues (Appendix B). Assays were always done in duplicate. The incubation mixture used in standard assays for lipoprotein lipase activity is shown in Table l. The reaction was terminated by adding 5 mi of chloroform: methanol (2:1) directly into the assay flask and mixing 20 sec with a vortex. After a 10 min equilibration period, .4 mi of .5 N H2304 was added and thecontents mixed twice more at 10 min intervals. The flask contents were transferred to a test tube, capped and allowed to stand overnight at 4°C. The tubes were centrifuged for 10 min at 2000 r.p.m. in a clinical centrifuge and the top layer (2 mt) 35 transferred to a second tube (using disposable pasteur pipettes) with- out disturbing the interface layer. The top layer was re-extracted by adding 3 mt of chloroform, mixing 20 sec, and equilibrating 10 min. The tubes were centrifuged for 10 min at 900 x g and .5 mi of the top layer mixed with 10 mt of scintillation fluid (Appendix A) for counting in a liquid scintillation counter (Nuclear Chicago). TABLE l.--IN VITRO ASSAY SYSTEM FOR BOVINE ADIPOSE AND MAMMARY LIPOPROTEIN LIPASEa Component Quantity mt Substrate Premix ' 0.6 Serum or 3% BSA-Saline 0.15 Heparin or .025 N NH4OH 0.05 .025 N NH40H 0.10 Homogenateb 0.10 aComponents added to 25 mt glass-stoppered flasks, incubated at 37°C for 60 min under air in a Dubnoff Metabolic Shaker. bHomogenate added after other components had been preincubated for 20 min at 37°C. Appropriate serum blanks (3% BSA-Saline instead of serum) were run with each assay. Blank values were subtracted from each estimate 36 of enzyme activity to remove residual lipase effects. Recovery of 14C-glycerol added to this system was 22.99%, if allowed to set over- night, yielding a recovery factor of 4.35. A sample calculation is shown below: a) CPM/serum flask minus CPM/saline blank = net CPM/flask. b) Net CPM/flask + CPM/uMole* = umoles/flask. *CPM/umole was calculated from CPM/10 pt premix minus back- ground CPM divided by nmoles in premix. 0.6 mi of premix contains 10 umoles of triolein plus a few hundredths umoles depending upon the specific activity of 3H—glyceryl trioleate. Therefore, 10 pt contains 0.01667 pmoles. c) umoles/flask times recovery factor times dilution factor times time factor = pmoles glycerol released hr‘1 9 tissue' . Lipolytic activity toward a serum activated triglyceride substrate, triolein, is referred to as lipoprotein lipase activity. The assay was linear with respect to tissue concentration. Linearity with time through 60 min was observed with adipose tissue LPL activity (Figure 1) but not with mammary tissue LPL activity (Figure 2). Mammary tissue LPL activity showed an early lag phase with a subsequent increase in the rate of glycerol release. This type of curve is suggestive of an initial buildup of monoglycerides with subsequent release of free glycerol. One of the major products of hydrolysis by LPL purified from postheparin plasma is apparently 2-monoglyceride (Fielding, 1972). Miller and Smith (1973) have observed an initial lag phase and attri- buted it to interaction of protein(s) with the surface film of a lipid 37 Fig. l.--Lipoprotein lipase activity in homogenates of adipose tissue at different incubation times. Conditions of assay were as described in Table 1 except incubation time was varied as indicated. Similar results were obtained in a similar trial with tissue from two other cows. Fig. 2.--Lipoprotein lipase activity in homogenates of mammary tissue at different incubation times. Conditions of assay were as described in Table 1 except incubation time was varied as indicated. Similar results were obtained in a similar trial with tissue from three other cows. Changing the substrate preincubation time from 20 min to 30 min gave similar results. -1 LPL ACTIVITY umoles gm tissue LPL ACTIVITY umoles 100 mg protein‘1 10 N 2o 38 ADIPOSE TISSUE [3 - Cow l x - Cow 2 INCUBATION TIME (Min) Figure l MAMMARY TISSUE A‘- l l 1 l l l 40 60 INCUBATION TIME (Min) _1 1 Figure 2 39 monolayer. Thus, the lag phase in our system could be due to insuffi- cient emulsification. However, this seems unlikely since the lag phase was not corrected by changing preincubation time from 20 to 30 min. In order to reduce the influence of the initial lag phase (< 10 min) all assays were run for 60 min. It is realized that this violates one of the rules for a valid assay; however, it is believed that this assay is quite suitable for detection of biological differences as reported herein. This view is supported by the fact that our findings with re- spect to LPL activity during various physiological states is in line with other reports (Hamosh et al., 1970; Askew et al., 1970; Robinson and Wing, 1970). Experimental Procedures Experiment T]: Effect of Induced Lactation on Enzymic Activity in Primiparous Bovines: Fetuses were removed by caesarean section in nine primiparous Holstein heifers at 180 days (six heifers) or 260 days (three heifers) gestation. Stage of gestation was determined from breeding dates and verified by rectal palpation. Mesenteric adipose tissue was sampled immediately after the fetus was removed (Shirley et al., 1973). Mammary tissue was sampled by biopsy (Oxender et al., 1971) approximately one hour after the fetus was removed. Following a l4-day lactation period, animals were sacrificed and samples of 40 mesenteric adipose and mammary tissue taken. Daily milk production was monitored during the lactation period. Live body weights and mammary gland weights were taken on the day of slaughter (Shirley et al., 1973). The ration consisted of 2.5 kg of a 16% protein concentrate, 7 kg of alfalfa hay and corn silage ad libitum before surgery. After surgery the ration was 7 kg of 16% protein concentrate plus alfalfa hay and corn silage ad libitum. Experiment T2: Enzymic Activity during various lactational states in multiparous bovines: Biopsy samples of mammary tissue and shoulder subcutaneous adipose tissue were taken at 49, 21, 14, 8, and 2 days prepartum and 7, 14, 28, 60, 120, 180, 240, and 300 days post— partum from eight multiparous Holstein cows. Each cow was bi0psied four or five times. The quarter of the udder sampled has a negligible effect on enzyme activity determination in the mammary gland (Askew et al., 1970). A complete outline of sampling dates relative to par- turition, number of samples per cow and how and where they were ob- tained is contained in Table 2. The animals were fed a normal herd ration consisting of a 16% protein concentrate, hay, and corn Silage in amounts required for maintenance and milk production during lactation. During the non— lactating period they were maintained on pasture with some additional 41 TABLE 2.--EXPERIMENT T2 SAMPLING DATESa Cows Samples 10W '51 1072 832 827 2095 2221 2520 Days Postpartum -49 RF -21 RF -14 RR 14 LR LF LF LF LF 28 RF LR LR LR 60 RF RF RF 120 RR RR RR 180 LF LF LF 240 LR LR LR 300 RF RF RF aAdipose tissue and mammary gland were sampled simultaneously. Mammary samples are identified as to the quarter they were taken from i.e. RF = Right front quarter, RR = Right rear, LF = Left front, LR = left rear, SL = Sampled immediately following slaughter. 42 hay or silage. Two weeks prior to parturition, they were challenge fed grain at the rate of .5 kg increments per day until they reached an in- take of 7 to 8 kg/day at parturition. After parturition, the amount of grain was increased until the individual cow reached maximum production or maximum intake. During lactation, adjustments in the amount of grain were made in accordance with declining milk production. Studies on Plasma Lipolytic Activity Plasma Sample Collection and Treatment Blood samples were collected from the jugular and mammary veins with a 10 mt disposable syringe equipped with a 20 gauge needle. Imme- diately after collection, the needle was removed and the whole blood injected into a test tube containing potassium oxalate (2 mg/mt whole blood). Samples were transported to the laboratory in an ice bath, immediately centrifuged at 900 x g for 20 min at 4°C. Plasma was re- moved with a pasteur pipette, placed in a plastic vial and stored at -40°C until assayed. Under these conditions, plasma lipolytic activity is stable for at least 2 months in our hands and up to 6 months as re- ported by others (Fielding, 1970). 43 Postheparin Plasma Samples Sodium heparin (5000 IU/cow) was injected into the jugular vein and blood samples collected at 5, 10, and 15 minute post injection. In some studies, samples were taken only 5 and 10 min or 10 and 15 min postinjection in lactating and non—lactating cows. Maximum postheparin plasma lipolytic activity was reached at approximately 10 min in pre- partum and 5 min in postpartum cows (Appendix C). A standard dose of 5000 IU of heparin was used even though dose response may be dependent on body weight. Mammary Arterio-Venous Technique To estimate that portion of plasma lipolytic activity contri- buted by the mammary gland, samples of both jugular and subcutaneous abdominal (mammary) vein plasma were obtained. The contribution by the mammary gland was determined by subtracting the lipolytic activity of jugular venous plasma from that of mammary venous plasma. This method assumes that jugular activity is representative of systemic contribution prior to entry into the mammary gland circulatory system. The mean lipolytic activity in Six paired samples of jugular vein ' plasma and plasma from the external pudic artery just prior to entry into the mammary gland agreed within 10 per cent. 44 Plasma Lipolytic Activity Assay Plasma lipolytic activity was assayed as described for tissue LPL activity with the following exceptions: l) 2.5 mi of serum from a lactating cow was added to the substrate premix prior to the sonication step, 2) the 20 min preincubation step prior to addition of enzyme was eliminated, 3) two concentrations of undiluted plasma (.2 and .3 ml) in duplicate were routinely included in each assay, 4) heparin was not routinely added to the assay mixture, and 5) an enzyme blank (3% BSA vice plasma) was included in duplicate in all assays to increase accur- acy of estimates of specific enzyme activity. A sample assay is out- lined in Table 3. This assay was linear with plasma concentrations TABLE 3.—-ASSAY FOR PLASMA TRIGLYCERIDE LIPASEa Component Quantity mil Substrate Premix .5 Plasma Sample .0, .2, or .3 3% bovine serum albumin—Saline .5, .3, or .2 aComponents added to 25 m1 flasks and incubated at 37°C for 60 min under air in a Dubnoff Metabolic Shaker. 45 used herein. The ratio of the activity obtained with .2 mt plasma and the activity obtained with .3 mi of plasma was .99 i .05 (n = 43 pairs) The assay was essentially linear with time through 60 min for both pre- heparin lipolytic activity (Figure 3) and postheparin lipolytic ac- tivity (Figure 4). Experimental Procedures Experiment P1: Plasma Lipolytic Activity vs Milk Fat Produc- tion: Six Holstein cows between 60 and 120 days in lactation ranging in milk production from approximately 11 kg/day to 40 kg/day were used. Individual milk and milk fat production were determined the evening and morning immediately prior to treatment and the evening immediately following treatment. Blood samples from the jugular and mammary veins were collected prior to and 5, 10, and 15 min after heparin (5000 units/cow) injection into the jugular vein. Samples were collected between 9 and 10:30 A.M. which was 4 to 6 hr after milking. Samples were handled as previously outlined. Feed consumption data was ob- tained from all cows and approximate "net energy” intake calculated (Appendix 0). Live body weights were measured on two consecutive days and the average used in calculating estimated net energy requirements (Appendix D). 46 Fig. 3.--Plasma lipolytic activity at different incubation times. Conditions of assay were described in Table 3 except incubation time was varied as indicated. Similar results were obtained with plasma from two other cows. Similar results were also obtained when the substrate premix was preincubated for 20 min prior to enzyme addition. LIPOLYTIC ACTIVITY 100 umoles mt plasma-1 10 47 PREHEPARIN PLASMA INCUBATION TIME (Min) Figure 3 48 Fig. 4.--Postheparin plasma lipolytic activity at different incubation times. Conditions of assay were as described in Table 3 except incubation time was varied as indicated. Similar results were obtained with postheparin plasma from two other cows. Similar results were also obtained when the substrate premix was preincubated for 20 min prior to enzyme addition. LIPOLYTIC ACTIVITY umoles m2 plasma" _a o 49 POSTHEPARIN PLASMA 4. 1. ///’/////+ a A i i J I 10 20 4o 50 INCUBATION TIME (Min) Figure 4 50 Experiment P2: Plasma Lipolytic Activity vs Adipose and Mammary Tissue Enzymic Activities: Samples of mammary tissue were obtained from eight Holstein cows in various lactational states. Tissue samples were taken either by surgical biopsy or following slaughter. In some cases, as many as four biopsy samples were taken from the mammary gland of an individual cow. When this was done, a different quarter was used each time. Lipoprotein lipase activity varies less than 10% among quarters (Askew et al., 1971). Animals were always-hand milked and jugular and mammary venous plasma samples taken within 1 hr prior to biopsy or slaughter. Milk and milk fat production data were obtained for the evening and morning milkings immediately prior to biopsy or slaughter. Experiment P3: Effect of Initiation of Lactation on Plasma Lipolytic Activity: Jugular and mammary venous blood was sampled from each of six Holstein cows on days seven and three prepartum (based on predicted calving date) and days three and seven postpartum. Following initial sampling, heparin was injected into the jugular vein and a second and third sample of jugular and mammary venous blood taken. Since predicted calving date and actual calving date did not always coincide, seven additional cows were used to fill in the pre- partum sampling dates and provide additional postpartum samples. 51 Experiment P4: Effect of Time after Milking on Plasma Lipo- lytic Activity: Four cows between 50 and 80 days in lactation receiv- ing the same diet were initially used to determine if milking affected lipolytic activity in mammary venous plasma. The animals were housed and milked separately from the herd.{ Mammary veing blood samples (10 ml per sample) were taken prior to the evening milking (3 P.M.), within one minute and at 10, 20, 30, 60, and 240 minutes after milking. Jugular vein plasma samples were taken prior to and within one minute after milking to determine prolactin response. Milk and milk fat pro- duction data were obtained for the morning and evening milkings on the day of the experiment. Plasma triglyceride lipase activity was mea- sured in mammary vein samples. Prolactin was measured in jugular vein plasma as outlined by Koprowski and Tucker (1971). Data from the initial four cows was insufficient to draw a conclusion and the milking procedure used resulted in decreased milk production during the experiment. Thus a second group of four cows was obtained for further observations. The second experiment was con- ducted as previously outlined with the following exceptions: a) the cows were milked in the parlor with the rest of the herd; b) jugular vein cannulas were installed three days prior to the experiment to facilitate sample collection and decrease the degree of animal excita- tion; and c) one minute and 20 minute post-milking samples of mammary 52 venous plasma were not obtained since the initial experiment indicated that samples taken at 10, 30, 60, and 240 minutes postmilking would provide ample observations. Other Methods Protein was determined by the method of Lowry et a1. (1951). Milk samples were tested for butterfat by the Babcock method. Plasma prolactin was assayed as outlined by Koprowski and Tucker (1971). Dry matter content of mammarystissue was determined by drying to a constant weight in a forced air oven at 85°C. Scheffe‘s test for significant . differences between means with heterogeneous variance and unequal 1 numbers was used for statistical analysis (Gill, 1971). RESULTS AND DISCUSSION Results of these studies are divided into two categories: 1) an investigation of enzyme activities in bovine adipose and mammary tissues during various lactational states, and 2) an investigation of plasma lipolytic activity with particular emphasis on its desirability as a measure of the mammary glands ability to clear triglyceride fatty acids from the blood. Results of both studies will be related to some extent in this section. Tissue Studies Introduction Induction of lactation is accompanied by a redistribution of lipid from adipose stores to the mammary gland. This redistribution phenomenon could be accomplished by differential regulation of blood fat uptake by the two tissues. The mechanism of blood fat uptake by adipose and mammary tissue is quite similar; fatty acids are hydrolyzed from blood triglycerides at the capillary endothelium and approximately 53 54 two-thirds of the fatty acids are reesterified into triglycerides within the cell (Scow et al., 1972). Both lipoprotein lipase (LPL) (Robinson and Wing, 1970) and glyceride synthesis (GS) (Shapiro, 1965) have been implicated as regulators of blood triglyceride fatty acid uptake by extrahepatic tissues. Investigation of the effect of lactation on these two enzyme systems in both adipose and mammary tissue should provide insight into their relative ability to perform a regulatory function and their involvement in the lipid redistribution phenomenon. In other words, does redistribution occur due to an increase in the mammary glands ability to clear blood triglycerides; a decreaSe in the ability of adipose tissue to remove triglycerides from the blood; or a combi- nation thereof. Two major experiments were undertaken to investigate these suppositions. In the initial experiment (Experiment T1), samples of adipose and mammary tissues were obtained from primiparous heifers to ascertain the effect of change in lactational state on adipose and mammary tissue LPL and GS attiVities. In a followup experiment (Ex- periment T2) biopsy samples of mammary and shoulder subcutaneous adi- pose tissue were taken at prepartum and postpartum from multiparous cows. The purpose here was to study the regulatory capability of LPL and GS with respect to both adipose and mammary tissue. 55 Experiment T1 General: Daily milk production during the 5 days before slaughter ranged from 3.5 to 11.5 kg. Average milk production was 5.9 kg and 10.4 kg for the 180— and 260-day heifers. Body weights before slaughter ranged from 342 kg to 399 kg, averaging 370 kg. The average mammary gland weights were 9.1 kg and 10.9 kg for the 180- and 260-day heifers. There were no significant correlations between enzyme activity and milk production, gland weight, or live body weight (Shirley et al., 1973). Enzyme data essentially were alike for both groups of heifers; thus, they were grouped and discussed together. Protein and dry matter: Dry matter content decreased from 20 per cent in prepartum tissues to 18 per cent in postpartum tissue to which oxytocin had been added to aid in removal of residual milk. Protein concentration followed the same pattern, decreasing from 130 i 13 mg/g tissue to 62 i 3 mg/g tissue. Approximately 56 per cent of this difference in protein can be attributed to the difference in dry matter of the tissues. Mean protein concentration of adipose tissue was 8 and 7 mg/g tissue at surgery and 14 days after caesarean section. Lipoprotein lipase and glyceride synthesis: LPL, which serves to deliver triglyceride fatty acids from blood to mammary cells, and GS, the process by which incoming fatty acids are incorporated into 56 triglycerides within the cell, increased 94 and 6-fold with onset of lactation (Table 4). Mammary LPL activity was augmented further by addition of heparin to the assay system (Table 5). Heparin stimulated LPL activity in both nonlactating and lactating tissue; however, lipo- protein lipase from nonlactating tissue Showed a greater response (P = .15) to heparin than did LPL from lactating tissue. A similar response was noted in tiSsue from multiparous cows although the re- sponse to heparin prepartum appeared to be somewhat related to time relative to parturition (Appendix C). TABLE 4.--LIPID METABOLISM IN PRIMIPAROUS BOVINE MAMMARY AND ADIPOSE TISSUE. Lipoprotein Lipase Glyceride Synthesis —------umoles hr’1 10 mg tissue protein‘1 ------- Mammary Pregnanta .5 : .2C .02 i < .01 Lactatingb 46.9 i 5.5 .12 i < .01 Adipose Pregnanta 12.0 i 3.0 1.2 i < 4} Lactatingb 8.0 i 1.0 .2 i < .01 aPrimiparous heifers 180 or 260 days pregnant. bFourteen days after caesarean section. c Values are mean i standard error of mean, N = 9. 57 TABLE 5.--MAMMARY LPL RESPONSE TO HEPARIN.a Heparin Added Pregnant , Lactating units/mt Homog. ------------ % of normalb ............ .05 218C 112d .125 243e 122f aHeparin obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. bNo added heparin. C’d’e’fMeans Of a given variable with different letters are signifi- cantly different. c > d (P = .08), e > f (P = .15). Activities Of LPL and GS in adipose tissue decreased with onset 1 10 mg protein-1) of of lactation (Table 4). Activity (umoles FFA hr- LPL decreased (P > .05) from 12 to 8 while GS decreased (P < .05) from 1.2 to .2 in adipose tissue from pregnant and lactating heifers. These results support the contention that an increase in mam- mary triglyceride uptake ability and a decrease in adipose tissue up- take ability are both involved in the redistribution of lipid at in- duction of lactation. The large increase in activity of mammary LPL relative to GS is suggestive of a regulatory role for LPL. With re- spect to adipose tissue, GS appears to be more sensitive to a change in lactational state than does LPL. 58 Experiment T2 Mammary gland: The capability Of the mammary gland to syn- thesize milk components is dependent on several factors, one of which is the enzymic complement Of the gland. One category of enzymes ap- pears to be constitutive in that their activity is relatively indepen- dent Of lactational state of the gland; the other category appears to be inducible since their activity is drastically affected by change in lactational state (Baldwin et al., 1966; Mellenberger et al., 1973). The inducible category apparently contains the regulatory enzymes such as lactose synthetase (Brew, 1969) and acetyl-COA carboxylase (Smith et al., 1966). To be considered regulatory to milk production an en- zyme should be sensitive to changes in lactational intensity and its activity should change prior to overt changes in mammary physiology. To study regulatory capability Of LPL and GS, biopsy samples of adipose and mammary tissues were taken at 49, 21, 14, 7, and 2 days prepartum and 7, 14, 28, 60, 120, 180, 240, and 300 days postpartum from eight multiparous cows. The results of this study are graphed in Figure 5. Mammary LPL activity (umoles glycerol released hr-1 mg pro- ] i S.E.) increased 6 fold between 49 and 7 days prepartum then tein‘ increased sharply (.4 to 5.1) between 7 and 2 days prepartum. LPL activity then increased at a decreasing rate, reaching a maximum 59 .sowpe>semso use an ueesemwsses use A. use .eFY .—N| .muu mzea .o.m A .u_ ms._ A .n+ MN.N A .w- mm.m A .oom mm.m A .oum mm.F A .owp we._ A .omp mm.— A .ou ”F.N A .wm "use scepwsspses Ease mzeu Au ueemwpswue seesm use so mseeE me see msosee useusepm .m_ A .s_ .n A .m m— A .N- ”w_ A .oom mm A .oem “me A .om_ mm_ A .om_ mom A .wN uwee scepwespses seem mzeu he uewmwpseuw usesm use so msees use sow msosse useusepm .mFemseuez use muospez se uenwsom neu me uwzemme use: meeuw>wpoe memespszm mueseoz—m use mmesw— sympossosws .N ereH se uese— upso use ueFQEem smuu: mo seesess use use Aswos seem pe msowpe>semeo mo sesszz .ANH pseewswsxmv mFewsepez use muespez se ueuwsomeu me we; essueooss Fepsweweesxm .e_oxo _esoepeuue_ eumrssoo e mswssu memmspszm euwsmo>Fm use emeswr swepoesosw~ meme?“ aseEEeE mo mewpw>wposomso oso es uopsomossos ose NT use .upu .FNI .muu exec .N..A .e— No._ A .n mm. A .N- me. A .oom mm. A .osm mo.— A .ow_ mN. A .o~_ MN.P A .ou me. A .mm nose screwssuses Eoss maeu so uowmepsouw seesm esp so mseoe mu soe msosso useusepm .s. A .s_ mm A .5 an A .m- as. A .oom m_ A .osm mm A .om_ mm A .omp mp A .ou mm. A .wm nose sowpwseuseo Eosm mzeu An uowmwusoup seesm use so mseoe use sou msosso useuseum .mpewsopez use muospoz s? uonesom uou me uoxemme osoz mowuw>epoe memosusxm ouwsooxpm use omeoep sAvosoosws .N opoeh s? uosAF upso ose uopssem souu: so sopseso use use Aseos soeo Ae msowpe>somoo so sensoz .Amh Asoeesosxmv m—eesouez use muospoz s? uoowsomou me we: ossuoooss pepsoeesosxm .o—vo sowueuoep ouoessoo e mswssu memosusxm ouwsooxrm use omeowp swopossost ozmmwp omoswue mo mowuw>epo .05) was Ob- served between milk fat production and peak postheparin lipolytic activ- ity in mammary venous plasma (MS-05). These results suggest that mam- mary gland LPL is normally released in proportion to the amount of blood triglyceride fatty acids taken up. Thus, as the amount of milk fat produced increases so does the release of LPL into the blood stream which leaves less LPL bound to the capillary membrane at a given time resulting in an inverse relationship between heparin releasable LPL and normal milk fat output by the gland. This interpretation necessarily implies that lipolytic activity in mammary venous plasma is a suitable measure of LPL or LPL-like activity. Further evidence in support Of this interpretation was provided by the Observation that milk fat production the P.M. subsequent to sampling was affected by heparin injection. All cows showed an increase in milk fat output the P.M. following heparin administration. However, degree Of response (post P.M.-pre P.M.) was negatively correlated (r = -.64, P > .05) with lipolytic activity in preheparin mammary venous plasma and positively 67 TABLE 6.--CORRELATION OF PLASMA LIPOLYTIC ACTIVITIES1 WITH MILK AND MILK FAT PRODUCTIONZ Plasma Sample3 Parameter M0 J0 MO-JO M5 J5 M5-J5 ----------- Correlation Coefficients----------- Milk Production (kg) pre A.M. ‘ .54 .40 .54 .23 .36 -.54 day .52 .27 .54 .24 .33 -.39 Milk Fat Production (kg) pre A.M. .78 .46 .80 .42 .47 -.30 day .78 .40 .81 .22 .31 -.36 post P.M.-pre P.M. -.64 -.41 -.65 .09 -.03 .41 Milk Fat Concentration (%) pre A.M. .40 .28 .41 .01 -.03 .12 day .49 .15 .52 -.20 -.19 .02 post P.M.-pre P.M. -.15 .20 -.08 .45 .42 -.04 1Activity against a serum activated lipoprotein triglyceride substrate expressed as umoles glycerol released hr" 2 100 ml plasma'l. Pre A.M. dicates values obtained at the A.M. milking before sampling. indicates summation of pre P.M. and A.M. milk and milk fat production. 3Origin Of lipolytic activity values. activity in mammary venous plasma; J0 = total preheparin lipolytic activity in jugular venous plasma; Mo-JO = preheparin lipolytic activ- ity in mammary venous plasma minus that in jugular venous plasma (an indication Of mammary gland contribution). same source as M0, J0, and MO-JO except activity is peak postheparin lipolytic activity. Milk and milk fat production were measured the P.M. and A.M. prior to blood sampling and the P.M. after sampling for 6 cows. in- M0 = total preheparin lipolytic M5, J5, M5-J5 represents 68 correlated (r = .41, P > .05) with peak postheparin lipolytic activity in mammary venous plasma. These results would be expected since post- heparin plasma lipolytic activity was higher in cows producing smaller as compared to those producing larger amounts Of milk fat as indicated by the inverse relationship between postheparin lipolytic activity and milk fat production. This would result in an increase in-intra-blood triglyceride hydrolysis and subsequent increase in fatty acids avail— able for uptake by the gland without the need for prior hydrolysis at the capillary membrane by LPL. Collectively these data strongly sug- gest that the physiologically active LPL is that normally released into circulation and not that available for release by heparin. Effect of Mammary Gland Emptying on Lipolytic Activity in Mammary Venous Plasma The relationship between mammary venous lipolytic activity and milk fat production was further characterized by Observing the effect of milk removal on lipolytic activity. Samples of mammary venous plasma were Obtained from eight cows prior to and 10, 30, 60, and 240 min after milk removal. Milk and milk fat production were determined the P.M. and A.M. prior to sampling. Results (Table 7) indicate that the level of lipolytic activity and presumably the amount of LPL released from mammary tissue is not 69 TABLE 7.--EFFECT 0F MAMMARY GLAND EMPTYING ON LIPOLYTIC ACTIVITY IN MAMMARY VENOUS PLASMA.‘ Pre- Time Post Milking (min) Parameter . - Mliklng +1 ’ +10 +20 +30 +60 +240 Lipolytic Activity (M0)2 5.7 4.4 5.9 5.8 5.6 6.3 6.3 Correlation 3 Coefficients (r) .43 --- .28 --- .37 .49 .71 1Samples of mammary venous plasma were taken from 8 cows prior to milk- ing and 10, 30, 60, and 240 min after milking. Additional samples were taken at l and 20 min after milking from 4 Of the cows. 2Total’activity in mammary venous plasma expressed as umoles glycerol released hr"1 100 ml plasma‘i. 3Correlates were M0 plasma lipolytic activity and milk fat production (kg/day) the P.M. and A.M. prior to sampling. N = 8, r = .71 for significance at P < .05. uniform during the milking interval. The level of activity increased with time after milking to 60 min, leveling Off until at least 240 min post milking, and then apparently decreased as the gland filled with milk. Time of sampling relative to time of gland emptying dramatically affects the relationship between mammary venous plasma lipolytic ac- tivity and milk fat output (Table 7). During our initial study, sampling was done between 9 and 10:30 A.M. which was between 4 and 6 hr after milk removal. Insignificant (P > .05) correlation coefficients 70 were Obtained (mammary venous plasma lipolytic activity vs milk fat production) immediately prior to and 10, 30, and 60 min after milking in the present study. The low correlation coefficients observed within the first hour after milking is primarily due to the inconsistency in plasma lipolytic activity response to milk removal. Lipolytic activity in mammary venous plasma increased post milking in four cows while it decreased or did not change in the other four cows. However, by 240 min post milking activity had returned to a level equal to or higher than that observed immediately pre-milking. The reason for these post milking fluctuations in activity is possibly a result of hormone(s) released due to milking. Since prolactin is released during milking (Koprowski and Tucker, 1971), it was thought that it might be involved. However, all cows showed a positive prolactin response to milking (Table 8), thus should have responded in the same direction unless an antagonistic hormone, possibly norepinephrine, was released in greater than normal amounts due to stress of bleeding. Results Of studies with two cows in which the jugular vein, external pudic artery, and mammary vein were equipped with indwelling cannulas indicated that infusion of prolactin into either the jugular vein or external pudic artery depressed mammary venous plasma lipolytic activity (Figure 7). Little or no effect was Observed on lipolytic activity in plasma from the jugular vein or external pudic artery. Collectively these data 71 TABLE 8.--PROLACTIN RESPONSE TO MILKING STIMULUSa Cow Sampling Time 1065 1072 1074 1128 1140 1189 1195 1196 ----------- Serum Prolactin-Nonograms mt‘1---——------- Pre Milking 44 36 40 41 25 17 28 31 Post Milking 46 48 64 75 63 116 117 158 aRadioimmunoreactive prolactin assayed in duplicate in serum samples taken from the jugular vein prior to preparation of the cow for milk- ing and within one min after milking machine was removed from the cow. Show that lipolytic activity in mammary venous plasma is affected by gland emptying and that this effect may be due to prolactin under non- stress conditions. A consistent decrease in lipolytic activity level would be in line with reports Of decreased glyceride uptake by the mammary gland during the early post milking period (Shaw and Petersen, 1940). Relationship between Plasma Lipolytic Activity and Energy Status Postheparin plasma lipolytic activity is depressed during fasting and returns to prefasting levels following refeeding. TO test the effect of energy status on mammary contribution to plasma 72 .eEmeFo msoso> sepzmuu s? >Aw>wpoe 1-1.1:: mesmeFQ Fewsouse oeuus Fessooxo sw >AA>PAoe illuiii mesme—s Feesopse ovuss Fessopxo s? xpw>wpoe muses eEmer muoso> xseeeee se zue>epoe iii in: mesmer msoso> aseEEeE s? auw>wpoe ilioili ”AAA>AAoe owA>FoowF so mossom .xsouse owuss Fessopxo osp opsw uopoomsw sAAoepossiifim zoo .seo> seesmsu osA oAse.ueromsw swuoeFossiummOF zoo .AFeAserz use muosuoz sw uoowsomou me uozemme we: >Aw>wpoe owpzpoowp esmeps .soepoouse Ame mv smpoeposs sopee use osomoo esopse owuss Fessopxo esp use mswo> xseEEeE use sersmou osp Eosm uoswepoo osoz eEme—s so mo—ssem .sowpoousw swpoe—oss oe >Aw>wpoe owes—osw— esmeyo so omsosmos Pesossoeii.m .mws 73 N os:m_s szszv zoseoeezs sees esse mu om ma 0 mal mm we on ma 0 mai omi mu: 4 d n q d n d d 1 q q q q n W o e o . c - a . 1 u g 1 III-‘4‘ /~ DQ‘ - [I]. 4“ cl ‘11 . . . a. e . . . .... . . o o h . I a! \\ Ia — \\ “ III \ I u\\ . a II:.\\ a; \\ B . a ’ \\ — \ D n o a - \\ II a L o \ In - . n \O o u OfIIIIIlI . 0. \\ I, . a \ . . o\\ \ a!” a . 0 — 0 ' _ .n...- . a ' ' ' n c ""I- s . .. Nu o y-0 I sowpoousH swpoe_oss sowpoonsH swooeFoss s _m 300 mwofi zoo O O H H O H N O ,4 [-0w OOl [-4H SBLOWH-AIIAIIOV OIIATOdIl VWSVld I_Tm OOT 1-1q sanmn ALIAILOV OILATOdIT VWSVTJ 74 lipolytic activity net energy (NE) intake (based on feed consumption data) and net energy required for body maintenance and milk production were calculated for six individual cows. The difference between energy intake and energy required was used as an index of energy status (Appendix D). Coefficients of correlation are given in Table 9. The negative correlation (r = -.32) between mammary venous plasma lipolytic activity (M ) and energy status suggest that high energy diets might have an O'JO adverse effect on the contribution of blood fat to milk fat. This rela- tionship may partially explain the depressed milk fat production asso- ciated with high grain-restricted roughage diets (Van Soest, 1963) which provides the animal excess energy, particularly in the form Of glucose (Jorgensen et al., 1965; Storry and Rook, 1966). The slight positive correlation (r a .17) Observed between milk fat output (kg/day) and energy status does not negate the above supposition but merely in- dicates that milk fat production is not entirely dependent on fatty acids from plasma and that an increase in de nOVO synthesis within the mammary gland may occur during luxury energy intake. Peak postheparin lipolytic activity in mammary venous plasma (MS-J5) was positively correlated (r = .67) with energy status. This supports the negative correlation Observed between MO-J0 lipolytic activity and energy status in that a decreased normal release of 75 TABLE 9.--CORRELATION OF ENERGY STATUSa WITH MILK FAT PRODUCTION AND PLASMA LIPOLYTIC ACTIVITIES b c c Milk fat % increase in Correlates MO-J0 M5 J5 (kg/day) M5 over M0 NE intake .06 .39 -—- -—— NET - NErbd -.32 .67 .17 .61 a . . .__ Energy status 15 defined here as the difference between net energy consumed and net energy required. hr = .8 for significance at P < .05. CMO-JO indicates mammary contribution to preheparin plasma lipolytic activity. M5-J5 indicates mammary contribution to postheparin plasma lipolytic activity. dNEi = net energy intake, NEr = net energy required. mammary LPL into circulation results in an increase in the amount avail- able for release by heparin. Another way to approach the amount Of lipolytic activity released by heparin is in terms Of the per cent in- crease relative to the M0 level. This approach aids in decreasing variability due to individual cow differences. The use of this value instead Of M -J values had little effect on the correlation between 5 5 postheparin lipolytic activity and energy status (Table 9). 76 Effect of Induction of Lactation on Plasma Lipolytic Activity Mammary tissue LPL activity increases several fold just prior to parturition in the bovine and continues to increase at a decreasing rate until near peak lactation. A working hypothesis is that the physiologically active LPL is bound to the luminal side of the capil- lary endothelium and is slowly released into the blood stream. In agreement with this hypothesis, we have found LPL activity in mammary venous plasma to be positively correlated with milk fat production (r = 0.8, P < .05). Thus, the availability of LPL for release into the plasma at induction Of lactation may be more meaningful in terms Of uptake of blood triglyceride fatty acids than total tissue activity. If the lipolytic activity in mammary venous plasma is due to release of LPL from the mammary gland, then appearance Of plasma lipo- lytic activity should be preceded by an increase in tissue activity of LPL. A corollary to this would be the inability of heparin to cause an increase in lipolytic activity in mammary venous plasma prior to an increase in tissue LPL activity. A second corollary is that heparin releasable lipolytic activity should appear before an increase in pre- heparin plasma lipolytic activity. To test this supposition, jugular and mammary venous blood samples were taken from six primiparous heifers at various times 77 during the interval from 17 days prepartum to 8 days postpartum. After initial sampling, heparin (10 IU/kg body wt) was injected into the jugular vein and a second and third sample of jugular and venous blood taken. Preheparin samples only were Obtained from an additional 7 heifers. Preheparin lipolytic activity was not detectable in either jugular or mammary venous plasma at 17 to 14 days prepartum (Table 10). TABLE lO.--PLASMA LIPOLYTIC ACTIVITY ACROSS PARTURITION IN PRIMIPAROUS HEIFERS. D Venous Plasma Source ays Postpartum Mammary Jugular Difference ----—------Lipolytic activity - umoles hr‘1 100 mt plasma‘1 ------------ -17 to -14 0(2)2 0(2) 0(2) -8 to -5 .08 s .04(5) .06 i .03(5) .03 i .03(5) -4 to -2 .22 i .07(10) .16 i .05(10) .06 i .06(10) Parturition .80 i .39(3) .22 : .18(3) .59 i 22(3) +2 to +4 4.70 i .64(5) .61 i .16(5) 4.09 s .60(5) +5 to +8 4.59 i .36(9) .62 i .10(9) 4.01,: .27(9) 1Days with similar values are grouped for conciseness. 2Values are given as mean i standard error. Numbers in parentheses are the number Of observations. Mean of postpartum values > parturi- tion values > prepartum values, P < .01. 78 Between 8 and 2 days prepartum, activity was detectable in small amounts and MO-J0 activity fluctuated between negative and positive values depending on the animal. The MO—J0 activity increased signif- icantly (P < .01) relative to prepartum levels on day of parturition and peaked out by 2 to 4 days postpartum. Postpartum values were significantly higher (P < .01) than values prepartum or values on day Of parturition. Mammary tissue LPL showed a major increase by 2 days prepartum (Figure 5). These data are in agreement with the stated supposition that prior increase in mammary tissue LPL activity is required for appearance of lipolytic activity in mammary venous plasma. In further support of this supposition are results obtained after heparin injection (Table 11). Peak postheparin lipolytic ac- tivity in mammary venous plasma (M—J) was negative between 14 and 7 days prepartum, indicating that no appreciable contribution was made by the mammary gland. Heparin releasable lipolytic activity in mam- mary venous plasma between 4 and 2 days prepartum was quite variable between animals; ranging from a positive 53 to a negative 9 units Of activity with a mean and standard error of 17 s 20. A large increase in postheparin lipolytic activity in mammary, jugular, and mammary minus jugular venous plasma was Observed on the day of parturition relative to prepartum activity. The ability Of heparin to increase plasma lipolytic activity was further enhanced by 2 to 4 days 79 TABLE 11.--PEAK POSTHEPARIN PLASMA LIPOLYTIC ACTIVITY1 ACROSS PARTURITION IN PRIMIPAROUS HEIFERS. Venous Plasma Source Days Postpartum Mammary Jugular Difference ----------- Lipolytic activity - umoles hr'1 100 mi plasma'1------------ -14 to -7 87 : 9(3)3 88 : 13(3) -o.3 : 4(3) -4 to -2 99 i 29(6) 82 1 21(6) 17 1 20(6) Parturition 375 s 60(2) 190 i 36(2) 175 i 14(2) +2 to +4 836 s 28(5) 692 i 30(5) 145 i 55(5) +5 to +8 806 s 99(8) 648 i 84(8) 159 1 31(8) 1Activity present after heparin (5000 IU/animal) injection in jugular vein. Samples taken 10 min postheparin in prepartum heifers and 5 min postheparin in postpartum heifers. 2Days with similar values grouped for conciseness. 3 . . Values represent mean i std error. Numbers in parenthes1s represent number Of Observations. postpartum; however, the mammary-jugular difference tended to decrease after parturition. This decline in mammary minus jugular activity was not significant and was possibly due to increased carry over of ac— tivity from mammary to jugular vein as a result Of an increased blood flow rate postpartum (Kjaersgaard, 1968). 80 Plasma Lipolytic Activity vs Adipose and Mammary Tissue LPL and GS Activity Mammary tissue LPL and GS activities are positively correlated with each other and with milk fat production (Askew et al., 1971). Similar relationships are reported elsewhere in this paper. Milk fat production was significantly correlated with mammary venous plasma lipolytic activity in this investigation. Thus, its relationship to mammary tissue LPL and GS activity was of considerable interest in determining its (plasma lipolytic activity) overall relevancy to blood triglyceride clearance by the mammary gland. Sampling dates relative to parturition and corresponding ac- tivities of plasma triglyceride lipase and mammary and adipose tissue LPL and G3 are shown in Appendix E. Correlation coefficients between the various parameters are in Tables 12 and 13. There appears to be little relationship between mammary tissue LPL activity and mammary venous plasma lipolytic activity if viewed prepartum or early post- partum (Table 12). The reason for the low correlation coefficients when the pre- and early post-partum states are considered alone is best explained by Figure 8. Mammary tissue LPL begins to increase around 7 days prepartum then increases at a decreasing rate to 60 days postpartum; while plasma lipolytic activity (M J0) does not increase 0- 81 TABLE 12.--RELATIONSHIP BETWEEN PLASMA LIPOLYTIC ACTIVITY AND MAMMARY TISSUE ENZYME ACTIVITIES AT VARIOUS LACTATIONAL STATES ‘Mammary Tissue Plasma Sample Characteristic Correlated with M0 J0 MO'JO Prepartum‘ (~30 to -2 days) LPL Activity .18(4) -.36(4) .25(4) GS Activity -.24(4) -.50(4) -.19(4) First Month of Lactation (7. 14, 28 days Post) LPL Activity -.12(6) -.70(6) .20(6) GS Activity -.46(6) .46(6) -.71(6) Complete Lactational Cycle (-30 to +300 days) LPL Activity .69°(13) .37(13) .69°(13) cs Activity .63b(l3) .7oa(13) .59b(13) aSignificant r value (P < .01), N = 13. bSignificant r value (P < .05), N = 13. 82 TABLE 13.--RELATIONSHIP BETWEEN PLASMA LIPOLYTIC ACTIVITY AND ADIPOSE TISSUE ENZYMIC ACTIVITIES AT VARIOUS LACTATIONAL STATES A‘ 4.. Adipose Tissue ‘ Plasma Sample Uizgjgy Characteristic Correlated with M0 J0 MO-JO LPL Prepartum (—30 to -2 days) LPL Activity -.90(4) -.95(4) -.86(4) .27(4) GS Activity -.44(4) -.07(4) -.48(4) ---- First Month of Lactation LPL Activity -.39(6) -.l4(6) -.36(6) -.60(6) GS Activity -.3l(6) .57(5) -.60(6) ---- Complete Lactation Cycle (-30 to +300 days) LPL Activity .73a(13) -.50(13) .7sa(13) -.50(l3) GS Activity .28(13) .10(13) .33(13) ---- aSignificant r value (P < .01), n = 13. 83 Fig. 8.--Pre- and postheparin lipolytic activity in mammary venous plasma and mammary tissue lipoprotein lipase activity across parturition. Preheparin plasma samples and mammary tissue samples were from the same coWs (Appendix E).. Postheparin samples were from primiparous heifers (Table 11). Experimental procedure was as described in Methods and Materials. Enzymic activities were assayed against a serum activated artificial triglyceride (triolein) emulsion. Plasma lipolytic activity is the activity in mammary venous plasma minus activity in jugular venous plasma. 84 O Preheparin plasma Postheparin plasma Mammary tissue .‘-b|"-."I|-"--'---'-l-""'.--"'.'"--...|"“-.‘x. /, i /. /. ”*7 Til/P P ‘U‘.- 'U'UDVI."OI‘."-‘L b V Di ’ I b b iP /’ /’ bi IF P F b P F P D O O O O O O O O O O 8 6 H 2 O 1. 2 3 Tu. 8 6 14 2 O 8 6 14 2 1.. . . . . 1.. 1.. 1.. 1. l _ _ . _ .Fiooso ozmmwp as oo_ so _-eEme_o as oo. Piss mo.o51v >AA>AAU< oAAXFooA. 9) Days Postpartum Figure 8 85 significantly until the day Of parturition and attains maximum levels within 3 days after induction of lactation. Therefore, over the short intervals of time included in the pre- and early postpartum period the relationship between the two is more like a precursor-product relation- ship than a direct in-phase relationship. In other words, it appears that the level of lipolytic activity in mammary venous plasma is depen- dent on a prior increase in LPL activity in mammary tissue. A precursor- product relationship is further indicated by the inability of heparin to increase M-J plasma lipolytic activity prior to the prepartum in- crease in activity Of mammary tissue LPL. When prepartum and early lactation data are Viewed as a unit in conjunction with data Obtained during late lactation (Table 12), a sig- nificant correlation (r = .69, P < .01) exist between MO-J0 lipolytic activity and LPL activity in the mammary gland. Mammary tissue GS and LPL activities were not significantly correlated in this study over a complete lactation. However, GS activity is significantly correlated with M -J 0 0 tivity in mammary venous plasma is in closer contact with tissue re- lipolytic activity. Thus, it appears that lipolytic ac- esterification Of incoming fatty acids than is the total tissue LPL; \ adding credence to the suppOsition that MO-J0 lipolytic activity more adequately defines physiological LPL activity than does total tissue i activity. The reason for the high positive correlation (r = .70, 86 P < .01) between jugular venous plasma lipolytic activity and mammary tissue glyceride synthesis activity is not readily apparent. This relationship might be due to a combination of factors: 1) J lipolytic 0 activity is normally quite low thus small real changes would appear large and 2) changes in J0 activity Often reflect changes in M0 activ- ity on a smaller scale. Relationships between plasma lipolytic activity and adipose tissue enzymic activities are depicted in Table 13. Primary consider- ation is directed toward the significantly negative correlation (r = -.73, P < .01) between MO-JO lipolytic activity and LPL activity of adipose tissue. Noteworthy also is the negative but nonsignificant correlation (r = -.50, P > .05) between LPL activity of mammary and adipose tissues. Taken together, these Observations concur with the reciprocal relationship concept between the two tissues; they also 0 O indicator of this reciprocal relationship than mammary tissue LPL suggest that plasma lipolytic activity (M —J ) may be a more sensitive activity. GENERAL DISCUSSION The destination of plasma triglyceridefatty acids is altered by physiological state and is apparently a function Of specific tissue need (Robbell and Scow, 1965). Diversion Of plasma triglycerides from adipose tissue to the mammary gland accompanies induction of lactation (Storry, 1970). The mechanism by which this phenomenon is effected is not clearly understood; however, differential regulation of trigly— ceride uptake by the two tissues appears to be a plausible effector. Results Of this investigation indicate that the ability of adipose tissue and the mammary gland to clear triglycerides from the .blood decreases and increases, respectively, with induction of lacta- tion. Similar results have been Observed in the rat (Homosh et al., 1970; Emery et al., 1971). The increase in mammary tissuelipoprotein lipase activity has been attributed to the Observed increase in pro- lactin at parturition (Scow, 1970). Evidence forprolactin stimula- tion Of mammary tissue LPL is available for the rabbit (Falconer and Fiddler, 1970) and rat (Shirley et al., 1972) but its effect on GS activity has not been Observed. The reason for the decrease in adi- pose LPL and GS activities is not clear, although it is quite possibly 87 88 due to hormonal changes associated with parturition (Convey, 1973). These changes in enzymic activity of adipose and mammary tissues are covert changes which could account for the redistribution of lipid from adipose to mammary tissue at induction of lactation. However, it appears that the negative effects of lactation on adipose enzymic activity can be overcome by luxury grain intake which is indicative of their previously reported sensitivity to energy status (Robinson and Wing, 1970) and insulin or glucose infusion (Rao and Hawkins, 1972). These results differ from those Of Hamosh et a1. (1970) in that they found adipose LPL to be low throughout lactation in the rat. This suggests the possibility that regulation of triglyceride uptake by adipose tissue in the rat and cow is mediated via different effec- tors. The 1ow LPL and GS activities in bovine adipose tissue during early lactation could be due to the associated negative energy balance. This would not be true for rats since the pups are tOO small during early lactation to provoke an energy insult. Lipoprotein lipase (LPL) is present in both adipocytes and vascular-stromal cells Of adipose tissue (Rodbell and Scow, 1965; Cunningham and Robinson, 1969) and presumably mammary tissue. Partial release of LPL from tissue can be accomplished by heparin perfusion (Rodbell and Scow, 1965), with a subsequent reduction in the tissues ability to hydrodyze a lipoprotein triglyceride substrate (Scow et al., 89 1972). Since heparin releasable LPL is presumably that LPL associated with or bound to the capillary membrane (HO et al., 1967) it appears that the functional site of LPL activity is at the capillary membrane. Strong evidence in favor of this View has been reported by Blanchette- Mackie and Scow (1971). Thus, the availability of LPL for release into the plasma may be more meaningful in terms of uptake of triglyceride fatty acids by a tissue than total tissue activity. Results of the experiments reported herein support the concept that the LPL available for release into the blood stream more accur- ately reflects the triglyceride uptake ability Of the tissue. In addi- tion, they carry the concept one step further in that they indicate that there are at least three pools of LPL. The functional pool is most accurately reflected by the LPL normally released into the blood. The three pools referred to include the intracellular LPL, the heparin releasable LPL, and the functionally released LPL. The relationship between these LPL pools appear to be Of the precursor—product type since the appearance of LPL-like activity in pre- or postheparin plasma is apparently dependent on a prior increase in tissue LPL activity (Figure 8). This type Of relationship is also indicated by the low positive correlation between preheparin plasma lipase and mammary tissue LPL just prior to and immediately following parturition. The two lipase activities are closely related only if the entire lactation 9O cycle is considered. Changes in preheparin plasma lipase activity are associated with Opposite changes in postheparin plasma lipase activity. Preheparin lipolytic activity in mammary venous plasma is positively correlated with milk fat production and negatively correlated with energy status while postheparin lipolytic activity is negatively corre- lated with milk fat production and positively correlated with energy status. In other words, an increase in the normal release rate of LPL 'in response to some physiological stimuli results in a decrease in the l_PL available for release by heparin while a decrease in the normal re- '1ease rate of LPL results in an increase in the LPL available for re- lease by heparin. In accord with this conceptual realtionship between pre- and postheparin lipolytic activity in mammary venous plasma is the rela- tionship between the two plasma lipolytic activities and the increase in milk fat output following heparin injection. Heparin injection in the jugular vein in the a.m. was followed by an increase in milk fat laroduction at the p.m. milking relative to milk fat output the p.m. prior to heparin injection. The degree of response in milk fat output to heparin injection was negatively correlated with lipolytic activity in preheparin mammary venous plasma but positively correlated with peak postheparin lipolytic activity in mammary venous plasma. The re- lease of LPL into the blood by heparin is associated with a decrease 91 in the concentration of plasma triglyceride and an increase in the concentration of plasma non-esterified fatty acids (NEFA) (LaRosa et al., 1971). An increase in plasma NEFA could result in a net up- take of NEFA by the mammary gland since net uptake is quite dependent on arterial concentration (Kronfeld, 1965). Postheparin plasma lipo- lytic activity was higher in those cows producing less fat; therefore, in theory, plasma NEFA's were higher. Thus, milk fat output in low fat cows could have been stimulated by an increase in the net uptake Of NEFA by the mammary gland. This interpretation is subject to crit- icism since only five to six hours elapsed between heparin injection and p.m. milking. This might not allow sufficient time for such an effect on milk fat output to occur. However, an increase in fatty acid availability for milk fat synthesis could possibly influence secretory rate. Another possibility is that heparin may exert some effect on mammary gland metabolism that is not apparent at this time. Collectively, these results lead to the hypothesis that the functional LPL is reflected by the lipolytic activity normally present in mammary venous plasma, that heparin releasable LPL is the immediate precursor of functional LPL, and that cellular LPL is the parent mate- rial for both pre- and postheparin lipolytic activity in mammary venous plasma. This concept of functional LPL is supported by data indicating the presence of both intra and extracellular LPL activity 92 in adipose tissue (Cunningham and Robinson, 1969). Additional support is provided by the data Of Schotz and Garfinkel (1972) indicating that two species of LPL are present in plasma. The plasma species appears to be identical to one of the tissue species. The suggestion that the physiologically active LPL is that LPL released by heparin (Borensztajn and Robinson, 1972) is an apprent conflict with the above hypothesis. However, this suggestion was based on the decrease Observed in tissue LPL activity following heparin perfusion and not on actual comparisons between pre- and postheparin plasma LPL activity and triglyceride uptake ability of the tissue. Thus, in essence there is no actual conflict since the proposed hypothesis would predict a decrease in tissue lipo- lytic activity against a lipoportein triglyceride substrate following exposure to heparin. Uptake Of plasma triglycerides by extrahepatic tissue involves prior hydrolysis by LPL (Scow, 1972) and subsequent reesterification within the cell (Shapiro, 1965). Since non-esterified fatty acids are negative modulators Of LPL activity (Gordon et al., 1953), a decrease in esterification rate could conceivably regulate triglyceride uptake by a tissue via end-product inhibition Of LPL. GS activity in mammary tissue homogenates was found to be posi- tively correlated with milk fat production in accordance with the results of others (Askew et al., 1971). However, it appeared to be 93 less sensitive to change in lactational state than either mammary tissue LPL activity or lipolytic activity in mammary venous plasma. The observation that GS activity was lower than tissue LPt during lactation could be taken to mean that GS was rate limiting with re- spect to triglyceride fatty acid uptake. However, this interpreta- tion is weakened by the Observed sensitivity of plasma lipolytic ac- tivity to lactational state, correlation with milk fat production, and low activity with respect to tissue GS activity. LPL (like) activity normally released into mammary venous plasma is a sensitive indicator Of the mammary glands ability to clear triglycerides from the blood. One of the major unsolved problems is the factor(s) controlling the transfer of LPL from the site of syn- thesis to the active cite at the capillary membrane. Another interest- ing investigation would be to determine if LPL activity in the venous plasma associated with other extrahepatic tissues is closely related to their triglyceride uptake ability. SUMMARY Lipoprotein lipase (LPL) and glyceride synthesis (GS) are con- Sidered essential for uptake of blood triglyceride fatty acids by ex- trahepatic tissues. Two experiments investigated the regulatory capa- bility of LPL and GS in bovine adipose and mammary tissues. Initially (Experiment T1) samples Of adipose and mammary tissues were Obtained from nine primiparous Holstein heifers 180 days (n = 6) or 260 days (n~= 3) in gestation and 14 days after induced (caesarean section) lactation. In the second experiment (12) biopsy samples of mammary and shoulder subcutaneous adipose tissue were taken at 49, 21, 14, 8, and 2 days prepartum and 7, 14, 28, 60, 120, 180, 240, and 300 days postpartum from eight multiparous Holstein cows. Mammary LPL activity (umoles fatty acids release hr.1 10 mg tissue protein-1) increased from .5 to 46.9 while adipose LPL de- creased (12 to 8) with onset of lactation (Experiment T1). Similarly, 1 10 mg tissue pro- GS activity (umoles palmitate incorporated hr- tein'1) increased 8 fold in mammary tissue and decreased 6 fold in adipose tissue.‘ In Experiment T2, mammary LPL activity (umoles gly- cerol released hr-1 100 mg tissue protein—1) increased 6 fold between 94 95 49 and 7 days prepartum, then increased sharply (.4 to 5.1) between 7 and 2 days prepartum, reached a maximum (83 s 12) at 120 days post- partum, and then decreased slowly until 280 days, postpartum (19 s 18). Mammary GS activity (umoles palmitate incorporated hr-1 100 mg tissue protein-1) decreased slightly between 49 and 8 days prepartum then increased 5 fold by 2 days prepartum and an additional 2 fold by 2 weeks postpartum after which it remained relatively constant until 280 days postpartum. Adipose tissue LPL and GS activities appeared to be more sensitive to energy status than to changes in lactational state (Experiment T Although activities of the adipose enzymes 2)' were variable, two discernible peaks occurred; one 2 days prior to parturition and one during mid-lactation (120 to 180 days). Both peaks correspond to luxury grain intake. Availability of LPL for release into plasma may represent up- take of triglyceride fatty acids by a tissue better than total tissue LPL activity since LPL catalyzes hydrolysis of blood triglycerides at the capillary membrane. Four experiments were conducted to ascertain the feasibility of using mammary venous plasma lipolytic activity (PLA), against a substrate Of triolein emulsion activated with serum as a measure of mammary clearance of triglycerides from blood. Pre- heparin and peak-postheparin PLA were measured and compared to milk fat production (an overt measure of plasma triglyceride utilization 96 by the mammary gland). In subsequent experiments, PLA was character- ized with respect to: 1) LPL and GS activities in adipose and mammary tissues, 2) energy status of the cow, 3) milking stimulus, and 4) changes in lactational state. Samples were taken simultaneously from the jugular and mammary veins and the difference in PLA between the two (M-J) was assumed to be the mammary gland contribution. Preheparin mammary PLA (M-J) was: 1) positively correlated (r = .8, p < .05) with milk fat production, manmary tissue LPL ac- tivity (r = .7, p < .01) and mammary tissue GS activity (r = .6, p < .05); 2) negatively correlated with energy status of the cow (r = -.3), adipose tissue LPL activity (r = -.7, p < .01) and adipose GS activity (r = -.3); and 3) sensitive to mammary gland emptying and prolactin injection (i.v.). Postheparin PLA in mammary venous plasma (M-J) was negatively correlated (r = —.4) with milk fat production and positively correlated with energy status of the cow (r = .7). Preheparin PLA was not detectable in either jugular or mammary venous plasma Of primiparous heifers at 17 or 14 days prepartum, but was detectable between 8 and 2 days prepartum. Mammary PLA (M-J) in- creased significantly (p < .01) on day of parturition and attained a maximum by 2 to 4 days postpartum. Postpartum PLA (M-J) was signifi- cantly higher (p < .01) than PLA prepartum or PLA on the day of par- turition. NO difference in heparin releasable PLA between jugular 97 and mammary venous plasma was Observed before 7 days prepartum. A mean positive M-J difference in postheparin PLA occurred by 4 days prepartum. Postheparin PLA in mammary, jugular, and mammary minus jugular venous plasma increased sharply at parturition followed by an additional increase by 2 to 4 days postpartum. Results of these experiments are consistent with the Views that (l) the redistribution Of lipid from adipose to mammary tissue at induction of lactation is due to a decrease in the uptake ability of adipose tissue and an increase in uptake ability of the mammary gland, (2) LPL released to mammary plasma is a more plausible regulator of triglyceride fatty acid uptake by the mammary gland than GS, (3) preheparin PLA in mammary venous plasma reflects the mammary glands ability to clear triglycerides from blood more accurately than tissue LPL activity or postheparin PLA, and (4) adipose tissue LPL and GA are more sensitive to energy status than lactational state. Results numbers 2 and 3 represent important new concepts. APPENDICES APPENDIX A COMPOSITION OF SCINTILLATION FLUID Component Quantity Paradioxane 770 mt Xylene 770 mt Absolute Ethanol 460 ml Napthalene 160 g PPO 10 g Dimethyl POPOP 0.1 g 98 APPENDIX B ADIPOSE AND MAMMARY LPL RESPONSE TO HEPARINa (EXPERIMENT T2) Mammary Adipose Lactationalb State -Heparin +Heparin Dif -Heparin +Heparin Dif -----LPL Activity - umoles hr“ 100 mg protein------ Days Prepartum -30 to -14 (3)C .17d .14 -0.03 6.08 7.03 +0.95 -7 (1) .40 4.30 +3.90 11.80 13.50 +1.80 -2 (2) 3.86 3.99 +0.10 9.50 12.77 +3.17 Days Postpartum 7 to 28 (5) 45.86 44.04 -l.82 2.20 2.15 -0.04 50 to 180 (5) 75.13 80.43 +5.30 5.15 5.30 +1.14 240 to 300 (5) 31.20 35.30 +5.10 1.75 2.10 +0.35 a.05 units of heparin added to the assay mixture prior to preincuba- tion. b Days with similar values are grouped for conciseness. cNumber of Observations included in the mean. dHeterogeneous variance between lactational states distracts from the usefulness of standard errors; therefore, they are not included. 99 100 Appendix C.--Temporal response of plasma lipolytic activity to intravenous injection of heparin. Heparin (5000 IU) was injected into the jugular vein and samples of plasma withdrawn simultaneously from the jugular and mammary vein at the time indicated. Plasma lipolytic activity was assayed as described in Methods and Materials. Prepartum samples were obtained three days before actual parturition. Postpartum samples were obtained l0 days after actual parturition. Source of lipolytic activity: -—-o-—- jugular venous plasma; ---x--- mammary venous plasma. Similar results were obtained with another cow. 101 m u t r a p t S 0 P ' 0 Prepartum X 0 0 “X"*----- -h 20 15 TIME (Min) 10 600 " P ImEmwpa as oo— Pug; mm—oE: >PH>HPQ< 4mg Appendix C APPENDIX D INDIVIDUAL cow DATA FROM EXPERIMENT P1 Cows Parameter - 923 824 950 1133 826 827 Body Weight (kg)a 591 756 75l 486 652 751 NE intake (Mcal) 15.0 46.3 44.2 16.9 34.1 22.4 NE required (Mcal) 25.5 32.9 35.9 27.2 23.1 19.7 NEi - NErd -10.5 +13.4 +8.3 -10.3 +11.0 +2.7 Milk Production (kg) Pre P.M. 10.4 16.0 16.1 12.1 6.4 5.4 Pre A.M. 14.1 21.0 22.7 17.7 12.7 5.6 Post P.M. 12.5 21.4 13.6 11.8 11.4 5.9 Mi1k Fat (kg) Pre P.M. .26 .37 .71 .36 .20 .20 Pre A.M. .42 .46 .70 .46 .37 .17 Post P.M. .45 .66 .61 .39 .40 .21 aMean of two consecutive day weights. bAverage caloric value of all dietary constituents consumed over a 5-day period prior to sampling. Caloric value (K cal) of feedstuff based on National Research cOuncil report (l97l). cDetermined by calculating energy required for maintenance of body weight and milk production, based on actual body weight, milk produc- tion, and fat test as prescribed by the National Research Council (l97l). dNEi = net energy intake, NEr = net energy required. 102 .mmmmgpcmcma :0 0L0 cams mcp :0 vmu:_ucw mcowwm>cmmno mo Lmnssz .mpnmuwpqaw :05; came mo Loccm vgwucmpm H cwmzo .Pucwmpoca mammwp me cop Pug: ummmoymc Pogmuzpm mw_oen FumEmmpg as oo_ 01;; ummmmch Fogmome mmposnm A000.NHH 0.0 0000.0 H 0._ A000.N H 00.0 A000.0_ H 00.0_ 0000. H 00.0 000+ A000. H 0A. 0000. H _N._ 000_.N H 00.0 00000 H 0N._0 0000. H 00.0 00+ A00“. H N0._ 0000._ H 00.0 000N.P H 00.__ 00000 H 00.00 0000. H 00.0 0+ A000. H 00., A000.“ H 00.0 ANVN.N H 00.0 0000 H 00.0 0000. H 00. N- 0,000.0 0000.__ A_000.0 00000. “0000.- 0- A0000. 00000. 0_000. 0A000_.- 00- 000 0000 000 0000 MMMMHWMW“ E00000 mammwh mmoawu< «20000 xgmsswz mammra ocuoz lumen made mocaom mFQEmm A00 02020000x00 mu oz< 0&0 mammHh >mHku<.oz< >HH>HPQ< QHH>0omH0