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University Microfilms International 300 N. ZEEB ROAD. ANN ARBOR, Ml 48106 18 BEDFORD ROW, LONDON WC1R 4EJ, ENGLAND 8106069

WHITEHURST, GARNETT BROOKS

LIPOGENESIS IN ADIPOSE TISSUE OF CATTLE

Jom State University PH.D. 1980

University Microfilms Int6rn sti 0 n al 300 N. Zeeb Road, Ann Arbor, MI 48106 Lipogenesis in. adipose tissue

from cattle

by

Garnett Brooks Whitehurst

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department; and Biophysics

Major: Biochemistry

Approved;

Signature was redacted for privacy.

Signature was redacted for privacy.

Signature was redacted for privacy.

Iowa State University Ames, Iowa

1980 11

TABLE OF CONTENTS

Page I. INTRODUCTION 1 II. LITERATURE REVIEW 4

A. Generation of Cytosolic Acetyl-CoA 4

B. Generation of Cytosolic NADPH 13

C. Regulation of Lipogenesis 19

D. Cellular and Enzymatic Changes in Adipose Tissue 27 E. Summary 31

III. FROM LACTATE IN GROWING CATTLE 33 A. Introduction 33

B. Materials and Methods 34

C. Results 40

D. Discussion 48

IV. OF PROPIONATE, GLUTAMATE, , LACTATE AND IN RUMINANT ADIPOSE TISSUES 55 A. Introduction 55 B. Materials and Methods 56

C. Results 59 D. Discussion 66

V. EXAMINATION OF A LOGNORMAL EQUATION TO DESCRIBE DISTRIBUTIONS OF DIAMETERS TO BOVINE ADIPOCYTES 71 A. Introduction 71

B. Materials and Methods 72

C. Results 77 Ill

Page D. Discussion 92

VI. DISCUSSION AND SUMMARY 95

A. Changes in Fatty Acid Synthesis and Lipogenic during Growth 95

B. The Citrate Cleavage Pathway and Fatty Acid Synthesis from Lactate 98

C. The Limitation for Glucose Use as a Fatty Acid Precursor 102

D. The Distribution of Adipocyte Sizes 104

VII. BIBLIOGRAPHY 106

VIII. ACKNOWLEDGEMENTS 122

IX. APPENDIX A 124

X. APPENDIX B 125

XI. APPENDIX C 126

XII. APPENDIX D 127

XIII. APPENDIX E 128

XIV. APPENDIX F 129 1

I. INTRODUCTION

The biological mechanisms responsible for the accumulation of in meat animals is of major scientific and economic interest. Approximately 4 billion pounds of fat are removed as wanted product from meat animals annually (Allen et al., 1976a, Hausman et al., 1979). This quantity of fat represents a sizable investment of crop resources, and considerable eco­ nomic savings could be realized if it could be reduced.

Consumers also have shown concern for the amount and type of fat in their diet. Pearson (1978) suggests that this con­ cern is in regard to the total quantity of fat in the diet and the potential link between consumption of saturated fatty acids, which are relatively high in animal products, and the develop­ ment of atherosclerosis. Although controversy still surrounds the role of animal fats in atherosclerotic lesion formation, consumers during the period from 1935 - 1973 have reduced intake of animal fat and replaced it with fat from sources (Pearson, 1978).

Adipose tissue is the site of storage in animals.

Factors that affect the amount of adipose tissue in an animal are its breed, plane of nutrition and management practices. In­ creased understanding of the role of these factors is needed if

the amount of fat associated with meat animals is to be reduced.

The quantity of lipid in adipose tissue is a function of

the number of adipocytes and the extent to which they are 2

"filled" with triglyceride, the principal stored lipid. To understand how adipose tissue grows, it is necessary then to understand the biochemical and physiological mechanisms regu­ lating both number and size of adipocytes, which is commonly referred to as cellularity.

The amount of triglycerides stored within the adipocyte represents the interaction of three factors: (1) the amount of fat that arrives at the adipocyte from dietary sources, (2) the amount of stored triglyceride that is hydrolyzed in situ for metabolism by the adipocyte itself or by other tissues and

(3) the amount of triglyceride synthesized de novo (Allen et al., 1976b).

The studies to be presented in the following reports will describe primarily the role of lactate as a precursor of fatty acids and hence triglycerides in bovine adipose tissue.

The specific objectives are:

1) To quantitate the utilization of lactate as a precursor of fatty acids in subcutaneous, intermuscular and intramuscular adipose tissues during growth of cattle. Lactate utilization for fatty acid synthesis will be compared with utilization of acetate in same tissues.

2) To relate the conversion of lactate to fatty acids to activities of key lipogenic and NADPH-generating enzymes to identify possible rate-limiting enzymes involved in conversion of lactate to fatty acids in bovine adipose tissues. 3

3) To determine if sufficient ATP-citrate lyase activity is present in bovine adipose tissues to support fatty acid synthesis from lactate via the citrate cleavage pathway.

4) To determine the role of the citrate cleavage pathway for cytosolic generation of acetyl-CoA glutamate, propionate and lactate will be compared as precursors of fatty acids. 5) To identify possible limiting steps in the utilization of glucose as a fatty acid precursor in bovine and ovine adi­ pose tissues.

6) To examine the use of a lognormal distribution func­ tion to describe size distributions of adipocyte populations in bovine adipose tissue. 4

II. LITERATURE REVIEW

The following review will deal with the generation of acetyl-CoA and TSIADPH for fatty acid synthesis in addition to the regulation of activities of acetyl-CoA carboxylase and fatty acid synthetase. Adipose tissue cellularity and devel­ opmental changes in adipose tissue also will be discussed. The term lipogenesis will be used interchangeably with fatty acid synthesis in the discussion. When in direct reference to a specific , the term activity will be used to indicate the specific activity as measured in tissue homogenates or particle-free supernatant preparations from a specific tissue.

A. Generation of Cytosolic Acetyl-CoA

Glucose is the principal precursor for fatty acid syn­ thesis in nonruminants (O'Hea and Leveille, 1969; Ballard and

Hanson, 1967a; Winegrad, 1965). Glucose is catabolized via the glycolytic pathway to pyruvate, which then is converted to ace- tyl-CoA by the mitochondrial complex. Because acetyl-CoA has only limited ability to transverse mito­ chondrial membranes (Spencer and Lowenstein, 1962), Spencer and Lowenstein (1966) suggested that citrate might serve to carry two carbon units across the inner mitochondrial membrane. Ci­ trate is formed from oxaloacetate and acetyl-CoA inside mito­ chondria by citrate synthetase (EC 4.1.3.7). Citrate can be cleaved in the cytosol to form oxaloacetate and acetyl-CoA by 5

ATP-citrate lyase (citrate cleavage enzyme; EC 4.1.3.7;

Bhaduri and Srere, 1962). Further , oxaloacetate from citrate cleavage can be utilized for NADPH generation by transhydroge- nation between NAD-malate dehydrogenase (EC 1.1.1.37) and NADP- malate dehydrogenase (decarboxylating, EC 1.1.1.40, malic enzyme. Young, Shrago and Lardy, 1964; Wise and Ball, 1964).

A problem with citrate cleavage is the possibility of depletion of mitochondrial oxaloacetate. Ballard and Hanson (1967b), however, found that rat adipose tissue contained sufficient (EC 6.4.1.1) activity to replenish oxalo­ acetate removed by citrate cleavage.

The citrate cleavage pathway for generation of cytosolic acetyl-CoA is illustrated in Figure 1. The sequence of re­ actions is the principal mechanism by which two carbon units derived from pyruvate or any pyruvate precursor are exchanged between mitochondria and cytosol. A key reaction in the path­ way is that catalyzed by ATP-citrate lyase (4).

Short-term regulation of enzymatic activity often is accomplished by covalent modifications of the enzyme, and often the covalent modification involves phosphorylation or dephos- phorylation of the enzyme (Krebs and Beavo, 1979). Lin and

Srere (1979) reported that ATP-citrate lyase underwent struc­ tural modifications by phosphorylation and observed that the structural behaved as a phosphate. The struc­ tural phosphate is distinctly different from the acid-labile, enzyme-bound phosphate associated with the catalytic cycle Figure 1. Citrate cleavage pathway. Legend: 1) pyruvate carboxylase, 2) pyruvate dehydrogenase, 3) citrate synthetase, 4) ATP-citrate lyase (citrate cleavage enzyme) 5) NAD-malate dehydrogenase, 6) NADP-malate dehydrogenase PYRUVATE

NADPH+H

NADP

PYRUVATE MALATE NAD ATP

NADH+H

OXALOACETATE OXALOACETATE

ATP CITRATE CITRATE

CYTOSOL 7

(Lin and Srere, 1979), Phosphorylation of the enzyme was not observed to alter the of the enzyme (Lin and Srere, 1979).

Ramakrishna and Benjamin (1979) have suggested that a phosphorylated upon insulin action in rat adipocytes is ATP- citrate lyase. Janski et al. (1979) have found that glucagon also stimulates phosphorylation (structural) of ATP-citrate lyase in rat hepatocytes. Therefore, ATP-citrate lyase is modified covalently by phosphorylation in response to hormonal stimuli; however, the role of phosphorylation of ATP-citrate lyase remains to be fully elucidated.

Activity of ATP-citrate lyase is altered by physiological situations that affect lipogenesis. Activity of the enzyme in rat liver is decreased by fasting and can be increased above normal by refeeding a diet high in glucose (Kornacker and

Lowenstein, 1965). Activity of ATP-citrate lyase is lower in liver of diabetic rats than in liver of normal rats and can be increased in liver of both normal and diabetic rats by insulin administration or feeding a high diet (Kornacker and

Lowenstein, 1964). Activity of the enzyme in rat mammary gland increases after parturition and remains high until weaning when activity decreases again (Howanitz and Levy, 1965). The specif­ ic activity of ATP-citrate lyase is greater in liver of fetal rats than in liver taken from neonatal rats (Ballard and Hanson, 1967c). 8

A regulatory role for ATP-citrate lyase in controlling rates of lipogenesis was suggested by Kornacker and Lowenstein

(1965). Studies by Foster and Srere (1968) suggest that the role, however, is not that of a primary regulator. Fatty acid synthesis from citrate and acetate in rat liver markedly in­ creases after 12 hours of refeeding following fasting, but ac­ tivity of ATP-citrate lyase does not change from fasting values. During fasting, fatty acid synthesis decreases mark­ edly while activity of ATP-citrate lyase remains unchanged

(Srere and Foster, 1967). Fatty acid synthetic rates from citrate cannot be altered in -free preparations of liver of fasted rats by addition of purified enzyme even though the added enzyme is still active (Foster and Srere, 1968). These results suggest that the primary regulation of lipogenesis in nonruminants occurs at some reaction other than that catalyzed by ATP-citrate lyase.

Ruminants, in contrast to nonruminants, absorb little glucose from the digestive tract (Elsden and Phillipson, 1948) and therefore must synthesize their glucose requirement from products of fermentation. Because they must synthesize glucose for body functions, it would seem desirable that rumi­ nants would spare glucose from fat synthesis. This concept has been supported by studies of Kleiber et al. (1955), Hardwick

(1963, 1964) and Ballard and Hanson (1967a). 9

Hardwick (1964) suggested that goat mammary gland was unable to cleave citrate derived from glucose and observed low activity of ATP-citrate lyase. In studies of glucose and ace­ tate metabolism by rat liver and adipose tissue, and sheep and cow livers and adipose tissues, Ballard and Hanson (1967a) ob­ served that glucose utilization for fatty acid synthesis by sheep and cow adipose tissues and livers was much less than that observed in rat adipose tissue and liver. They also ob­ served much lower activities of enzymes of the citrate cleav­ age pathway in sheep and cow adipose tissues and livers. In particular, in sheep and cow livers, activities of NADP-malate dehydrogenase and ATP-citrate lyase were less than 5% of the activity in rat liver. In sheep and cow adipose tissues, spe­ cific activities of NADP-malate dehydrogenase were 20% of those observed in rat adipose tissues. Specific activities of ATP- citrate lyase in sheep and cow adipose tissues were 20% and

45%, respectively, of that found in rat adipose tissue. Ac­ tivity of pyruvate carboxylase in sheep and cow adipose tissues was 1% and 10%, respectively, of that in rat adipose tissue.

Thus, it seemed that ruminant liver and adipose tissue had low activity of the citrate cleavage pathway.

ATP-citrate lyase activity is much greater in fetal liver of ruminants than in liver of mature ruminants (Hanson and

Ballard, 1968). Hanson and Ballard (1968) observed 20-fold greater specific activity of ATP-citrate lyase in liver of 10 fetal oxen compared with liver of adult oxen. Fatty acid synthesis from glucose, aspartate and glutamate were greater in liver slices of fetal oxen than from same substrates in liver slices of mature oxen. Both aspartate and glutamate can be utilized only for fatty acid synthesis via citrate cleavage (Leveille and Hanson, 1966a, 1966b). The studies of

Hanson and Ballard (1968) led to the suggestion that the in­ ability of mature ruminants to utilize glucose for fatty acid synthesis was because of lack of sufficient citrate cleavage pathway activity.

Whether citrate cleavage activity adapts to dietary con­ ditions or altered physiological states in adult ruminants is unclear. Ballard et al. (1972) observed two-fold increases in conversion of glucose to fatty acids in liver and adipose tissue of sheep by feeding -rich diets or by intra­

venous infusion of glucose. Also, activity of ATP-citrate

lyase increased in adipose tissue and liver with carbohydrate

feeding and intravenous infusion of glucose. Adipose tissue showed the greater response to infusion of glucose or to carbo­

hydrate feeding (Ballard et al., 1972). Baldwin and Ronning

(1966), however, observed no significant difference in ATP-

citrate lyase activity in adipose tissue when calves were fed high-carbohydrate or high-fat diets; greater activity was ob­

served on the high-carbohydrate diet. Young et al. (1969) also

observed no differences in ATP-citrate lyase activity in liver, adipose tissue or rumen mucosa of steers fed either hay or con­ centrate diets. Fasting for 8 days had no effect on specific activity of ATP-citrate lyase in same tissues of cows (Young et al., 1969). It is not clear from these studies whether

ATP-citrate lyase has a regulatory role in controlling rates of fatty acid synthesis in ruminant tissues.

The investigations by Ballard and Hanson (1967a) suggested that acetate and not glucose was a more important precursor for lipogenesis in ruminants. Investigations by Hood et al. (197 2) and Ingle et al. (1972) also support the finding that acetate is a more important precursor for lipogenesis than glucose.

Propionate is a poor precursor for lipogenesis in ruminants (Hood et al., 1972).

The first reaction in the utilization of acetate for lipogenesis is catalyzed by acetyl-CoA synthetase (EC 6.2.1.1).

Greater activity of the enzyme is present in ruminant adipose tissue and liver than in nonruminant adipose tissue and liver

(Ballard and Hanson, 1967a). Activity of acetyl-CoA synthetase in rat liver is reduced by fasting (Kornacker and Lowenstein,

1965; Murthy and Steiner, 1972) and is lower in liver of al-

loxan-induced diabetic rats than in liver of normal rats

(Murthy and Steiner, 1972). Activity of the enzyme is decreased

in ovine adipose tissue by fasting, but decreases are not ob­

served until after 48 hours of fasting while decreases in fatty acid synthesis from acetate are observed within 24 hours (Ingle 12 et al., 1973). This observation by Ingle et al. (1973) sug­ gests that acetyl-CoA synthetase does not have a primary regu­ latory role in fatty acid synthesis in ruminants. Recent studies have suggested that substrates other than acetate can be used for lipogenesis in ruminants. Whitehurst et al. (1978) observed that lactate could be utilized for fatty acid synthesis in vitro by bovine adipose tissue at rates simi­ lar to those for acetate in the presence of glucose. Synthetic rates from pyruvate were greater than those from glucose. Prior (1978) found that lactate could be used 40% to 70% as well as acetate for carcass lipid formation in sheep, depending on amount of energy intake. Fasting decreased rates of fatty acid sythesis from lactate in bovine adipose tissue, and rates of fatty acid synthesis from lactate increased above prefasting values after refeeding for 15 days (Prior and Jacobson, 1979a).

Prior and Jacobson (1979b) found that pyruvate could be uti­ lized at greater rates than acetate in the absence of glucose in vitro by bovine adipose tissue; Prior and Jacobson (1979c) have reported greater rates of utilization of pyruvate than glucose for fatty acid synthesis by bovine adipose tissue.

The significance of the results of Whitehurst et al. (1978),

Prior (1978) and Prior and Jacobson (1979a, b, c) is that their findings challenge previous explanations for the low rates of fatty acid synthesis from glucose in ruminant tissues. Low activity of the citrate cleavage pathway had previously been 13 accepted as the explanation for low rates of glucose utili­ zation for fatty acid synthesis.

Observations that both lactate and pyruvate are used at greater rates than glucose for lipogenesis indicate that some other metabolic step in the pathway of glucose utilization for lipogenesis is limiting. Several possibilities exist.

Glucose uptake or phosphorylation might be limiting because liver of ruminants has lower specific activity of glucokinase compared with liver of nonruminants (Ballard and Oliver, 1964).

A reaction of the glycolytic pathway also might be limiting.

Smith (1975) observed that rabbit adipose tissue utilized glu­ cose at lower rates than pyruvate and suggested that this was because of low pyruvate kinase activity.

Therefore, it seems well-established that acetyl-CoA for fatty acid synthesis is formed from either citrate or free acetate. In ruminants, little glucose is catabolized to acetyl-

CoA, and acetate is a more important precursor for acetyl-CoA formation. Commonly accepted explanations for the low rates of fatty acid synthesis from glucose in ruminants need to be re­ investigated because of recent findings on lactate use.

B. Generation of Cytosolic NADPH

Fatty acid synthesis requires reducing equivalents in the form of NADPH in addition to acetyl-CoA (Langdon, 1955). NADP-

malate dehydrogenase, NADP-isocitrate dehydrogenase (EC 1.1.1. 42), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 14

6-phosphogluconate dehydrogenase (EC 1.1.1.44) are potential

"generators" of NADPH. The two latter enzymes are part of the pentose phosphate pathway (Figure 2).

Because of the low activity of NADP-malate dehydrogenase found in ruminant tissues (Ballard and Hanson, 1967a, c; Bauman et al., 1970), malic enzyme contributes little to cytosolic

NADPH generation in ruminants. Baldwin et al. (1976) estimated that the activity of this enzyme contributes 4% of total cy­ tosolic NADPH generated in adipose tissue of lactating dairy cattle. It should be noted, however, that activity of NADP- malate dehydrogenase markedly increases when ruminants are fed high-carbohydrate diets (Young et al., 1969; Ballard et al.,

1972). Thus, NADPH generation via this reaction may be more important in ruminants fed high-energy rations. Bauman (1976) has suggested that the reaction catalyzed

by NADP-isocitrate dehydrogenase is an important source of cytosolic NADPH in ruminants. Total activity of the enzyme is greater than that for glucose-6-phosphate dehydrogenase in

adipose tissue (Ingle et al., 1972, 1973; Baldwin et al., 1973).

The enzyme also is quite active in lactating mammary tissue of

sheep (Gumaa et al., 1973) and cows (Bauman et al., 1970).

Specific activity of the enzyme in adipose tissue decreases in

response to fasting (Pothoven and Beitz, 1975; Ingle et al., 1973) and increases in response to feeding of high-energy diets

(Baldwin and Ronning, 1966; Opstvedt et al., 1967). Baldwin et al. (1976) estimated the contribution to NADPH generation by Figure 2. Pentose phosphate pathway. Legend; 1) Glucose-6-phosphate dehydrogenase, 2) D-glucono-6-lactone hydrolyase (EC 3.1.1.17), 3) 6-Phosphogluconate-dehydrogenase, 4) Ribosephosphate (EC 5.1.3.1), 5) Ribulosephosphate-3-epimerase (EC 5.3.1.9). 15b

GLUCOSE-6-PHOSPHATE L NADP"^ I ® l^-^NADPH+H"^ e-PHOSPHO-GLUCONO-LACTONE A

-H2O +H20 © t 6-PHOSPHO-GLUCONATE NADP" © NADPH+H+ RIBULOSE-5-PHOSPHATE

RIBOSE-5-PHOSPHATE XYLULOSE-5-PHOSPHATE Figure 2. Continued. 6) Glycoaldehydetransferase (Transketolase; EC 2.2.1.1), 7) Dihydroxyacetonetransferase (Translaldolase; EC 2.2.1.2), 8) Glucose- 6-phosphate isomerase (EC 5.3.1.9). -5-PHOSPHATE XYLULOSE-5-PHOSPHATE

©

SEDOHEPTULOSE-7-PHOSPHATE GLYCERALDEHYDE-3-PHOSPHATE

FRUCTOSE-6-PHOSPHATE ERYTROSE-4-PHOSPHATE

GLUCOSE-6-PHOSPHATE GLYCERALDEHYDE XYLULOSE-5-PHOSPHATE 3-PHOSPHATE 17

NADP-isocitrate dehydrogenase in adipose tissue of lactating dairy cattle to be 32% of total NADPH needed for lipogenesis.

Pentose phosphate cycle activity is believed the major source of NADPH for lipogenesis. The two enzymes of the cycle responsible for NADPH generation are glucose-6-phosphate dehy­ drogenase and 6-phosphogluconate dehydrogenase (Figure 2).

Flatt and Ball (1964) estimated from carbon balance studies that from 50% to 60% of the NADPH needed for lipogenesis in rat adipose tissue was generated by pentose phosphate cycle-

Rognstad and Katz (1966) also estimated that 50% to 60% of cytosolic NADPH was generated via pentose phosphate pathway in rat adipose tissue incubated in the presence of insulin. The contribution increased to 80% in the presence of epinephrine.

Baldwin et al. (1976) estimated from modelling studies that

64% of the NADPH needed for lipogenesis in adipose tissue of a lactating dairy cow was generated by the pentose phosphate pathway. As has been observed with other NADPH-generating enzymes, activities of glucose-6-phosphate dehydrogenase and

6-phosphogluconate dehydrogenase are increased by high-concen­ trate feeding (Young et al., 1969; Opstvedt et al., 1967) and decreased by fasting (Pothoven and Beitz, 1975). Measurement of the amount of glucose catabolized via pentose phosphate cycle can be accomplished several ways. Re­ covery of [^^C] from [1-^^C]-glucose and [6-^^C]-glucose in

COg, fatty acids, and other metabolic products can be 18 utilized (Katz and Wood, 1960). The recycling of label through the cycle necessitates estimation of major products of glucose metabolism instead of simply using ratios of [ 14 COg] recovered from [1-^^C]-glucose and [6-^'^C]-glucose. Another procedure is to isolate a product such as glycogen derived from the 14 phosphate pool produced from metabolism of [2- C]-glu­ cose (Landau and Katz, 1964). Recycling would result in sig­ nificant quantities of label appearing in carbon 1 and 3 of the glucose in the hexose phosphate pool (Wood and Katz, 1959) and hence label would appear in carbons 1 and 3 of the glucose residues of glycogen. Degradation of the glycogen and deter­ mination of the quantities of label appearing in each carbon of the glucose so formed can be utilized to estimate pentose

cycle metabolism (Landau et al., 1964). Using the glycogen

degradation method from [2- 14 C]-glucose. Landau and Katz (1964)

estimated that 13% of glucose occurred via pentose

cycle in rat adipose tissue in absence of insulin and 23% of glucose catabolism occurred via pentose cycle in the presence

of insulin. Similar results were obtained by Katz et al. (1966)

using specific yields of [^^COg], [^^C]-fatty acids and

glycerol from tissue incubations with [1-^^C]-glucose and

[6-^^C]-glucose. The results by Katz and coworkers indicate

that significant metabolism of glucose occurs via pentose phos­

phate pathway in adipose tissue and suggest that this pathway

is the major source of NADPH for fatty acid synthesis. 19

C. Regulation of Lipogenesis

1. Acetyl-CoA carboxylase

Gibson et al. (195 8) isolated two soluble enzyme fractions from pigeon liver capable of fatty acid synthesis and observed

that synthesis of fatty acids required ATP, bicarbonate, a

metal ion and NADPH. The observation of a bicarbonate require­ ment was significant in that it suggested that fatty acid synthesis did not occur via reversal of beta-oxidation. One of the protein fractions was found to catalyze carboxylation of acetyl-CoA to form malonyl-CoA (Wakil, 1958) and was later observed to contain protein-bound biotin (Wakil and Gibson,

1960). The second fraction was found to catalyze palmitate synthesis from malonyl-CoA that required the presence of

acetyl-CoA (Wakil and Ganguly, 1958). Ganguly (1960) proposed

that formation of malonyl-CoA was rate limiting for fatty acid

synthesis. Studies by Numa et al. (1961) supported this pro­ posal. Fatty acid synthesis from acetate in particle-free

supernatant fractions from either fasted or fed animals was

observed to correspond closely to activity of acetyl-CoA

carboxylase. In sheep and cattle, acetyl-CoA carboxylase

activity decreases with the same time course as fatty acid syn­

thesis during fasting and increases with the same time course

as fatty acid synthesis during refeeding (Ingle et al., 1973;

Pothoven and Beitz, 1975). Pothoven and Beitz reported a corre­

lation of .99 for specific activity of acetyl-CoA carboxylase 20 and rates of fatty acid synthesis from acetate in adipose tissue of cattle during fasting. Acetyl-CoA carboxylase has been purified from a number of sources: bovine adipose tissue (Moss et al., 1972), rabbit mammary gland (Manning et al., 1976; Hardie and Cohen, 1978), rat mammary gland (Miller and Levy, 1969) and chicken liver

(Gregolin et al., 1968a). The enzyme exists in an active polymeric form and in an inactive protomeric form (Gregolin et al., 1966a, b). The polymeric enzyme forms filamentous structures (Gregolin et al., 1966a; Kleinschmidt et al., 1966).

The protomeric form of the enzyme can be further dissociated into two subunits (Mackall and Lane, 1977) and has been found to contain one molecule of biotin per subunit (Mackall and

Lane, 1977; Tanabe et al., 1975). Studies with acetyl-CoA carboxylase of have supported a two-step reaction mechanism. The first step is the ATP dependent carboxylation of biotin at the I'-ureido N position (Guchhait et al., 1974a). This reaction is catalzed by the biotin carboxylase subunit of the E. coli carboxylase. The carboxylated biotin is located on the carboxybiotin car­ rier protein, also a subunit of the E. coli carboxylase system.

The second step of the catalytic mechanism is the transfer of carboxylate group from carboxybiotin carrier protein to ace- tyl-CoA catalyzed by carboxyltransferase subunit (Polakis et al., 1974). The E. coli carboxylase has been used to study the catalytic mechanism because of the ability of the subunits 21 to be individually isolated (Guchhait et al., 1974b), which has not been possible with animal carboxylases (Bloch, 1977).

Brady and Gurin (1952) observed that fatty acid synthesis from acetate in homogenates of pigeon liver was stimulated by citrate and isocitrate. The stimulation of fatty acid synthe­ sis by citrate later was found to be due to an increase in the activity of acetyl-CoA carboxylase (Martin and Vagelos, 1962;

Matsuhashi et al., 1962; Waite, 1962; Kallen and Lowenstein,

1962). Activation of carboxylase by citrate involves a change in sedimentation behavior (Vagelos, et al. 1963; Gregolin et al., 1966b). The change in sedimentation behavior corre­ sponds to a change from inactive protomeric enzyme to the active polymeric enzyme (Gregolin et al., 1966b). Isocitrate

(also an activator) increases the value but does not affect for acetyl-CoA (Ryder et al., 1967; Gregolin et al., 1968b).

Whether citrate is responsible for activation inside the cell has been difficult to determine. Spencer and Lowenstein

(1964) observed no changes in citrate content of rat liver due to fasting or fluoroacetate poisoning. Halestrap and Denton

(1974) observed no correlation between acetyl-CoA carboxylase activity and citrate concentrations in rat adipose tissue.

Goodridge (1973) did find a correlation between citrate con­ centration of isolated chick hepatocytes and fatty acid synthe­ sis. Watkins et al. (1977) found parallel time courses for 22 increases in intracellular citrate concentrations and fatty acid synthesis in chick hepatocytes, Meredith and Lane (1978) pointed out that previous studies had measured total citrate concentrations and devised a procedure for estimating cytosolic citrate concentration. The procedure involved perforation of plasma membranes with digitonin in the presence of an ATP trapping system (to reduce loss of citrate due to ATP-citrate lyase activity) and benzene tricarboxylic acid (an inhibitor of mitochondrial citrate transport; Robinson et al., 1970).

Using this procedure, 75% of cellular citrate was found to be cytosolic; glucagon and dibutyryl-cAMP reduced cytosolic and mitochondrial citrate concentrations. The effect of glucagon and dibutyryl-cAMP was reversed by pyruvate. Digitonin per­ foration of plasma membranes was observed to result in release of protomeric carboxylase, and the release of carboxylase activity could be increased by dibutyryl-cAMP and reduced by citrate (Meredith and Lane, 1978). Malonyl-CoA also stimulated release of carboxylase from digitonin-treated cells. Malonyl-

CoA depolymerizes carboxylase (Gregolin et al., 1966b, 1968b).

Thus, most studies do suggest that alterations in intracellular citrate concentrations have a significant role in regulation of carboxylase activity. In addition to regulation by citrate, acetyl-CoA carboxy­

lase activity also is inhibited by fatty acyl-CoA (Numa et al.,

1965; Goodridge, 1972). Also, fatty acid synthesis is 23 inhibited by fatty acyl-CoA (Porter and Long, 1958). Inhi­ bition of carboxylase by palmitoyl-CoA is competitive with respect to citrate and noncompetitive with respect to acetyl-

CoA, ATP and HCO^ (Numa et al., 1965). Palmitoyl-CoA causes depolyraerization of the enzyme (Numa et al., 1965), and in­ hibition by palmitoyl-CoA can be reversed by addition of al­ bumin, citrate or palmitoyl-carnitine (Goodridge, 1972). Interpretation of enzyme inhibitions by fatty acyl-CoA must be made with some reservation. Srere C1965) observed that citrate synthetase was inhibited by palmitoyl-CoA and that ci­ trate synthetase could be protected against inhibition by pal- mitoyl-CoA by addition of either oxaloacetate or bovine serum albumin. Srere (1965) suggested that palmitoyl-CoA was a natural dénaturant. Taketa and Pogell (1966) found that a number of enzymes could be inhibited by palmitoyl-CoA and sug­ gested that inhibition of enzyme activity by palmitoyl-CoA was because of its detergent properties. When enzymatic assays were conducted in the presence of albumin, Goodridge (1972), however, only observed inhibition of acetyl-CoA carboxylase by palmitoyl-CoA. Thus, it is not clear whether inhibition of carboxylase by palmitoyl-CoA in vitro is a specific effect upon acetyl-CoA carboxylase or a nonspecific detergent effect of the compound.

Fatty acyl-CoA concentrations are increased by fasting

(Bortz and Lynen, 1963; Tubbs and Garland, 1964; Goodridge, 1973) 24

and can be decreased by feeding glucose to fasted animals

(Goodridge, 1973). Incubation of rat adipose tissue with in­ sulin in the presence of glucose reduces fatty acyl-CoA concen­ trations greater than incubation with glucose alone {Halestrap and Denton, 1974). Incubation with insulin alone has no effect

on fatty acyl-CoA concentrations. Acetyl-CoA carboxylase ac­ tivity increases in rat adipose tissue after incubation with

glucose or glucose plus insulin (Halestrap and Denton, 1974).

Some investigators, however, do not find a correlation of fatty

acyl-CoA concentration and acetyl-CoA carboxylase activity

(Jacobs and Majerus, 1973). Thus, it seems that the physiolog­

ical role of fatty acyl-CoA in control of acetyl-CoA carboxy­

lase activity remains to be fully elucidated.

Phosphorylation and dephosphorylation may have a signif­

icant role in the hormonal control of acetyl-CoA carboxylase

activity. Carlson and Kim (1973) observed that the enzyme

could be phosphorylated and that the phosphorylated form was

partially inactivated. Carboxylase is inactivated by incuba-

tion with ATP and Mg 2+ (Greenspan and Lowenstein, 1968; Lee et al,, 1973). The y phosphate of ATP becomes covalently

bound to the enzyme (Carlson and Kim, 1974; Lee and Kim, 1977) ,

and increases in phosphate incorporation occur at low concen­

trations of citrate in the presence of cAMP (Lent et al., 1978) and by epinephrine treatment of rat adipocytes (Lee and Kim,

1978). Lee and Kim (1979) found that the enzyme phosphorylated 25 in absence of epinephrine was fully active, but the enzyme phosphorylated in the presence of epinephrine was inactivated.

The depolymerized forms of the enzyme occurring after phospho­ rylation in the presence of epinephrine cannot be repolymerized by citrate (Lee and Kim, 1978). Dephosphorylation of the enzyme that had been phosphorylated in the presence of epine­ phrine is enhanced by either or Mg^^ in the presence of n « Ca (Brownsey et al., 1979). The role of phosphorylation in the regulation of carboxylase activity remains to be fully elucidated.

Long-term regulation of acetyl-CoA carboxylase activity involves changes in the intracellular quantity of enzyme. Majerus and Kilburn (1969) found 5-fold increases in synthesis of the enzyme in liver after fat-free feeding of fasted rats.

Half lives of 48 hours were observed in fat-free fed rats and of 18 hours in fasted rats. In rats fed a 12% fat diet, deg­ radation rate was unaltered but synthetic rate was decreased.

Mackall and Lane (1977) also have suggested that changes in carboxylase activity in mammary gland during the lactation cycle are because of increases in enzyme synthesis. Nakanishi

and Numa (1970) found that synthesis of carboxylase in liver was decreased in fasted rats and in diabetic rats. Only fast­ ing, however, increased the rate of degradation. Therefore, it

seems that both alterations in synthesis and degradation of 26 carboxylase protein are involved in long-term adaptations to altered physiological states.

2. Fatty acid synthetase

Wakil (1958) described a protein fraction from pigeon liver that catalyzed formation of palmitate from malonyl-CoA. The protein required NADPH for activity, and acetyl-CoA en­ hanced the activity (Wakil and Ganguly, 1958). Propionyl-CoA and butyryl-CoA also could be utilized for fatty acid synthesis (Wakil and Ganguly, 195 8). The protein described by Wakil was the fatty acid synthetase complex, and much difficulty has been encountered in elucidating the molecular structure of the complex from mammalian sources (Bloch, 1977).

Fatty acid synthetase activity is subject to dietary and hormonal control (Burton et al., 1969). Fasting animals and alloxan-induced diabetic animals had lower specific activity of synthetase. Safflower oil feeding reduced activity of the enzyme (Wiegand et al., 1973).

Alterations in synthesis of fatty acid synthetase are responsible for changes in activity observed in changing phys­ iological states. Inhibitors of protein synthesis, puromycin and actinomycin D, prevent increases of activity due to refeed- ing fasted animals (Hicks et al., 1965). Craig et al. (1972) observed increases in synthesis of fatty acid synthetase after refeeding fasted animals, and feeding safflower oil reduced synthesis of synthetase protein (Flick et al., 1977). Changes 27 in cellular concentration of synthetase because of dietary alterations are caused by changes in content of cellular mes­ senger RNA (Zehner et al., 1977; Nepokroeff and Porter, 1978). Much accumulated evidence suggests that primary regulation of rate of fatty acid synthesis occurs at the reaction cata­ lyzed by acetyl-CoA carboxylase. Short-term regulation of acetyl-CoA carboxylase activity involves changes in intracellu­ lar concentrations of citrate and palmitoyl-CoA and in changes in the phosphorylation state of the enzyme. Long-term regu­ lation of lipogenesis is accomplished via changes in intra­ cellular content of acetyl-CoA carboxylase and fatty acid synthetase. There is little evidence to suggest that fatty acid synthetase has a primary role in regulation of lipogene­ sis; however, the quantity of intracellular synthetase is con­ trolled by diet and fasting.

D. Cellular and Enzymatic Changes in Adipose Tissue

Adipose tissue is a type of connective tissue that is composed of adipocytes (fat cells), intercellular spaces, nerves and vascular tissue. The tissue functions as an energy reservoir and an insulator and as a source of heat in the case of brown adipose tissue. Adipocytes are the most metabolically active cells in adipose tissue (Rodbell, 1964).

Increases in adipose tissue mass are due to increases in adipocyte number (hyperplasia), increases in adipocyte size

(hypertrophy) or both. Whether increases in fat cell number 28 can occur at any stage of growth is unclear. Hirsch and Han (1969) found that neither starvation or hypothalamic damage changed the number of adipocytes in rats. Faust et al. (1978), however, found that adipocyte number was increased by feeding high-fat or high-carbohydrate diets and that cell number did not decrease if rats were allowed to lose excess weight. Faust et al. (1978) suggested that attainment of some mean adipocyte size triggers development of new adipocytes. Klyde and Hirsch

(1979a) have suggested that there is a rapidly proliferating cell type closely associated with mature adipocytes which could be the adipocyte progenitor. The DNA of this cell type 3 was rapidly labelled with [ H]-thymidine in vivo or in vitro. 3 High fat feeding increased labelling by [ H]-thymidine (Klyde and Hirsch, 1979b). Thus, this rapidly proliferating cell may be responsible for dietary induced alteration in adipocyte number (Klyde and Hirsch, 1979b).

In meat animals, whether hyperplasia or hypertrophy is ob­

served depends on the depot considered. Hood and Allen (1975a) observed increases in cell number in cattle occurring at 14 months of age in interfasicular (intramuscular) fat, but in­ creases in cell number were not observed beyond 8 months of age in subcutaneous and perirenal adipose tissues. Garbutt et al. (1979) have found increases in cell number in perirectal adipose tissue of bull calves up to 1 year of age. In swine. 29

Lee and Kaufman (197 4a) observed increases in cell number in intramuscular adipose tissue until 24 weeks of age.

Activities of lipogenic enzymes also vary with increasing age or animal weight. However, the method of expression of data can influence results. When data were expressed as nmole utilized (units) per 100 mg of wet tissue; Hood and

Allen (1973a) observed decreases in activity of NADP-malate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phospho- gluconate dehydrogenase in perirenal and middle subcutaneous adipose tissues of swine; however, increases were observed when data were expressed as units per mg soluble protein or as units per 10^ cells. The increases observed were more pro­ nounced when data were expressed as units per 10^ cells com­ pared with units of mg protein.

In swine, Lee and Kaufman (1974b) observed increases in

activity of NADP-malate dehydrogenase in middle subcutaneous,

trapezius and semitendinosus adipose tissues of Duroc pigs

from 2 weeks to 24 weeks of age. In Hampshire pigs, increases

in activity of NADP-malate dehydrogenase occurred in trapezius

and semitendinosus adipose tissues from 2 weeks to 24 weeks of age. Activity of NADP-malate dehydrogenase increased from 2

weeks to 16 weeks and decreased from 16 weeks to 24 weeks in

middle subcutaneous adipose tissue of Hampshire pigs. Change

in activity of ATP-citrate lyase paralleled those of NADP- malate dehydrogenase in both breeds and in all three adipose 30 tissues (Lee and Kaufman, 1974b). Increases in NADPH gener­ ating ability with increasing age also have been observed in swine of Hampshire X Yorkshire breeds and Minnesota #3 X Minnesota #1 breeds increasing in weight, and Minnesota #3 X

Minnesota #1 generally had greater activities of NADPH gener­ ating enzymes (Hood and Allen, 1973b). Little alteration in activity of acetyl-CoA carboxylase was observed by Anderson and Kaufman (197 3) in adipose tissue of swine from 1 month to 10 months of age. Parr (1973) observed increases in activity of acetyl-CoA carboxylase in sheep from 8 weeks to 32 weeks of age.

Few studies concerning enzymatic or fatty acid synthetic changes during growth have been done with cattle. Pothoven and Beitz (1973) observed increases in activity of glucose-6- phosphate dehydrogenase in outer backfat, inner backfat, peri­ renal fat and omental fat of cattle growing from 278 kg to 528 kg. In all but perirenal adipose tissue, steers of 3 85-kg weight group had highest activity, but none of the changes with increasing weight was statistically significant. Their results were expressed as activity per mg protein. Fatty acid synthetic rates from acetate were greatest in the 2 85-kg weight

group, and subcutaneous adipose tissue had the greatesc syn­

thetic rate of fatty acids. Differences among breeds or types of cattle in fatty acid synthetic capacity and lipogenic enzyme activities in adipose 31 tissue have been observed. In general, cattle of dairy breeds have lower activities of NADPH generating enzymes (Hood and

Allen, 197b), and activity of acetyl-CoA carboxylase is lower in cattle of dairy breeds than cattle of common beef breeds

(Chakrabarty and Romans, 1972). Also, fatty acid synthetic rates are lower in dairy cattle breeds compared with beef cattle breeds (Chakrabarty and Romans, 1972). Scott and

Prior (1980) have observed lower activity of fatty acid synthe­ tase in subcutaneous and perirenal adipose tissues of large- type cattle (Charolais, Gelbvieh, Maine-Anjou and Brown Swiss

X Angus or Hereford) than in small-type cattle (Angus X Here­ ford). Acetyl-CoA carboxylase, glucose-6-phosphate dehydrogen­ ase and 6-phosphogluconate dehydrogenase activities were not different in either depot between the two types of cattle.

E. Summary

Acetate, not glucose, is the principal precursor for lipogenesis in ruminants. Because both lactate and pyruvate can be utilized at greater rates than glucose, it would appear that insufficiency in some reaction other than ATP-citrate

lyase accounts for the inability or ruminants to utilize glucose for fatty acid synthesis.

Acetyl-CoA carboxylase catalyzes the rate-determining step

in fatty acid biosynthesis. Alterations in the intracellular

concentration of citrate or palmitoyl (fatty acyl)-CoA or in the phosphorylation state of the enzyme change its activity and 32 either singly or in combination result in the short-term responses of fatty acid synthesis to hormonal stimuli. Long- term regulation of fatty acid synthesis involves changes in the intracellular content of involved in fatty acid synthesis. Synthesis of both acetyl-CoA carboxylase and fatty acid synthetase is decreased during fasting and is increased upon refeeding of fasted animals.

Adipose tissue increases in mass by either hyperplasia and hypertrophy during growth. In addition to changes in cellular size or number of fat cells in adipose tissue, fatty acid synthetic capacity and enzymatic capacity in adipose tissue vary during the maturing process of animals and between different breeds or types of animals. 33

III. FATTY ACID SYNTHESIS FROM LACTATE IN GROWING CATTLE

A. Introduction

Acetate, not glucose, is the principal precursor for fatty acids in ruminants (Ballard and Hanson, 1967a; Hood et al.,

1972; Ingle et al., 1972), and adipose tissue is the principal site of lipogenesis in ruminants (Ballard and Hanson, 1967a;

Hood et al., 1972. The inability of ruminants to utilize glu­ cose for fatty acid synthesis has been attributed to the low activity of the citrate cleavage pathway (Ballard and Hanson,

1967a). Recent investigations have demonstrated that lactate is a fatty acid precursor in ovine (Prior, 1978) and in bovine adipose tissues (Whitehurst et al., 1978; Prior and Jacobson,

1979b) and have challenged the explanation that low citrate cleavage pathway activity results in poor utilization of glu­ cose for lipogenesis in ruminant tissues (Ballard and Hanson,

1967a). No studies of lactate utilization for lipogenesis in cattle at several stages of growth and development have been

performed. The objective was to quantitate the use of lactate and ace­ tate for lipogenesis by subcutaneous, intermuscular and intra­ muscular adipose tissues from two frame sizes of cattle of sev­ eral different ages. To test the hypothesis that lactate con­

version to fatty acid requires citrate cleavage pathway, activ­

ity of ATP-citrate lyase was determined in the same depots.

Furthermore, activities of acetyl-CoA carboxylase and the 34 principal NADPH-generating enzymes in ruminants (Baldwin et al., 1976; Bauman, 1976) were measured to identify possible rate-limiting steps in lactate conversion to fatty acids.

B. Materials and Methods

1. Experimental design

Thirty-two crossbred steers, progeny of Limousin, Main-

Anjou, Angus and Simmental sires and crossbred cows, of simi­ lar birthdates were used. Cows were either two-way or three- way crosses among Angus, Hereford, Holstein and Brown Swiss breeds. Steers were allotted by frame size primarily according to their body weights at 180 days of age, adjusted on the basis of weight per day of age at weaning. Measurements of wither height and rump height also were used for allocation to frame- size groups.

Steers within the two size groups were assigned at random to four slaughter groups. Steers were housed outdoors in the same pen and fed ad libitum a ration on a dry-matter basis, consisting of 72% ground shelled corn, 22% alfalfa-brome hay- lage or oatlage and 6% pelleted 32%-protein supplement. At 11,

13, 17 and 19 months of age, eight randomly selected steers, four of each frame size, were slaughtered at Iowa State Univer­ sity's Meat Laboratory in Ames, Iowa. Steers were transported from their feedlot at the McNay Research Center at

Chariton, Iowa, to Ames, a distance of approximately 80 miles, about 20 hours before slaughter and housed in the Meat Science 35

Laboratory overniqht, where feed and water were provided.

2. Tissue sampling

Immediately after exsanguination, a portion of the right longissimus dorsi and associated fat corresponding to the 10th through 12th rib was removed. Subcutaneous adipose tissue over the 12th rib and intermuscular and intramuscular adipose tis­ sues of the longissimus muscle were removed and kept in 0.9%

NaCl solution at 37°C until assayed for fatty acid synthesis or until homogenization for enzymatic assays. Subcutaneous and intermuscular adipose tissues were obtained within 10 minutes after removing logissimus muscle, and an additional 20 minutes generally were needed to dissect intramuscular adipose tissue from the longissimus muscle.

3. Fatty acid synthesis assay

A 100 mg to 200 mg section of adipose tissue was incubated for 2 hours at 37°C in 3 ml of Krebs-Ringer bicarbonate buffer 2+ (Ca -free; Laser, 1961) containing either 150 ymoles of L(+)- sodium lactate plus 15 ymoles of glucose and 1 of L(+)- sodium-[U-^'^C]-lactate (New England Nuclear, Boston, Massachu­ setts) , or 75 ymoles of sodium acetate plus 15 ymoles of glucose and 1 viC^ of sodium-[l-^^C]-acetate (Amersham Sear le , Arlington

Heights, Illinois). Triplicate determinations were conducted for each adipose tissue sample and substrate combination. In­ cubations were terminated by addition of 0.5 ml of 1-5 N H2S0^. 36

Tissue sections incubated in the presence of served as controls. After termination of incubations, tissue sections were

separated from incubation media by filtering through glass wool and then washed with 0.9% NaCl solution. Lipids were

extracted from tissue overnight on a wrist-action shaker with 20 ml of chloroform:methanol (2:1 (v/v); Pothoven and Beitz,

1973). The resulting lipid extracts then were washed as

described by Folch et al. (1957) and evaporated before saponi­

fication for two hours at 85°C in 3% methanolic KOH. Fatty

acids were separated from saponified lipid extracts with hexanes (mixture of isomers of hexane) as described by Pothoven

and Beitz (1973) and, after evaporating hexanes extracts, the

fatty acids were assayed for radioactivity by liquid scintilla­

tion counting. Counting was performed with a Beckman Model LS-

8000 liquid scintillation spectrophotometer, and quenching was

corrected by external standard method.

4. Tissue homogenization

A weighed amount of adipose tissue was homogenized at 37°C

for 30 seconds in a buffer solution containing 0.25 M sucrose, 1 mM EDTA, 1 mM glutathione and 10 mM Tris-HCl, pH 7.4, with a Virtis Model 45 top-drive homogenizer. The ratio of homogeni­ zation buffer to tissue was 2:1 (v/w). Tissue homogenates were contrifuged at 15,000 x g for 20 minutes at 23°C. The solution

above the pellet and below the fatcake was removed, and the 37 volume was measured. Aliquots were utilized for enzymatic assays and protein determination. Protein concentration was determined as described by Lowry et al. (1951), using bovine serum albumin as a standard.

5. Enzyme assays

Acetyl-CoA carboxylase (EC 6.4.1.2) was assayed at 37°C

as described by Dakshinamurti and Desjardens (1969) as modi­ fied by Anderson et al. (1972). Final reagent concentrations were: 0.1 mM EDTA, 8 mM MgCl2/ 3 mM reduced glutathione, 5 mM ATP, 5 mM potassium citrate, 0.6 mg/ml bovine serum albumin

(Fraction V), 0.2 mM acetyl-CoA and 10 mM NaH^^CO^ (0.5 yC^/

ymole, Amersham Searle, Arlington Heights, Illinois) buffered with 50 mM Tris at pH 7.6. Bovine serum albumin was defatted

before use as described by Fain (1975). The total assay volume

was 1.0 ml and contained 0.6 ml of tissue homogenate. Assays were started by addition of NaH^^COg, ATP and acetyl-CoA after

a 30-minute preincubation with citrate at 37°C. Acetyl-CoA

and ATP were omitted in controls. Triplicate determinations were performed with each adipose tissue depot.

Activity of ATP-citrate lyase (EC 4.1.3.8) was determined at 23°C as described by Srere (1962). Final reagent concentra­

tions were 10 mM MgClg, 20 mM potassium citrate, 10 mM mercap- toethanol, 0.4 mM coenzyme A, 0.2 mM NADH, 500 units/ml malate

dehydrogenase (porcine heart; Sigma Chemical Company, St. Louis,

Missouri) and 10 mM ATP buffered with 10 mM Tris-HCl at pH 7.3. 38

The total volume was 1.0 ml and contained 0.2 ml of tissue homogenate from subcutaneous or intermuscular adipose tissue and 0.1 ml of tissue homogenate from intramuscular adipose tissue. Reaction was initiated by addition of ATP, and change in absorbance at 340 nm was followed with a Gilford Model 2000 recording spectrophotometer. All assays were conducted against a control with ATP and coenzyme A omitted.

Because of low activity of ATP-citrate lyase in bovine adipose tissue, assay for ATP-citrate lyase activity was monitored by measuring the same activity in a 100,000x g super­ natant preparation from rat liver prepared as described earlier for homogenization of adipose tissue except that a 15,000 x g supernatant was centrifuged at 100,000 x g for 90 minutes at

0°C to 4°C with a Beckman Model L-2 ultracentrifuge fitted with a 40.3 rotor. Homogenization of rat liver and centrifuga- tion at 15,000 x g were conducted at 0°C to 4°C. The 100,000

X g supernatant fraction of rat liver was stored at -20°C and remained active for at least 18 months.

Activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.

49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) were assayed by the double substrate procedure of Clock and McLean

(1953). Increases in absorbance at 340 nm were followed as described for assay of ATP-citrate lyase. Final assay volume of 2.4 ml contained 0.05 ml of tissue homogenate from either subcutaneous or intermuscular adipose tissue and 0.1 ml of 39 tissue homogenate from intramuscular adipose tissue. Reactions were started by addition of 0.1 ml of 50 mM glucose-6-phosphate or 0.1 ml of 50 mM glucose-6-phosphate plus 50 mM of 6-phospho- gluconate.

Activity of NADP-isocitrate dehydrogenase was assayed as described by Ochoa (1955). Volumes of tissue homogenate from specific adipose tissues used for assay were identical to those used for assay of glucose-6-phosphate dehydrogenase and 6-phos- phogluconate dehydrogenase. Substrate (dl-isocitrate) was omitted from controls, and increases in absorbance at 340 nm were determined as indicated previously.

6. Adipose tissue cellularity

A weighed (approximately 200 mg) section of adipose tissue was fixed with 2% OsO^ in 50 mM collidine chloride buffer so­ lution at pH 7.6 for 72 hours (Hirsch and Gallian, 1968). The fixed adipose tissues then were treated with 8 M urea to solu- bilize connective tissue as described by Etherton et al. (1977).

The fixed, urea-treated adipocytes were freed by washing with

0.9% NaCl solution containing 0.01% of Triton X-100 through a

250 ym nylon mesh filter and collected on a 20 ym nylon mesh filter. The freed adipocytes then were washed with 0.9% NaCl solution into a preweighed 400 ml plastic beaker; the volume was increased to approximately 350 ml with the NaCl solution, and the beaker was reweighed.

Number of adipocytes was determined by counting ten 0.5 ml 40 aliquots with a Coulter Counter Model ZB in the linear mode.

A 400 iJm aperture was used. Base channel threshold was set to exclude material less than 20 ym in diameter. The total number

of adipocytes per gram of tissue then was calculated by multi­

plying the average of the ten 0.5 ml counts by difference in

weight of full and empty beaker and dividing by density of

0.9% NaCl solution (1.0053 g/ml) and by tissue weight.

7. Statistical analysis

Data were analyzed as a completely randomized design

(Snedecor and Cochran, 1967). Sums of squares for each vari­

able were partitioned according to frame size and age effects and according to frame size by age interactions. Multivariate

tests for an overall frame size effect within an adipose tissue depot were conducted as described by Morrison (1967). Pair

comparisons of means within an adipose tissue depot were per­

formed with Student's t-test using the pooled error mean- square for each measurement within an adipose tissue depot from

the analysis of variance.

C. Results

An objective of this experiment was to quantitate lipid

synthesis in two frame sizes of cattle during growth. Statisti­ cally significant differences between frame sizes of steers,

however, were not detected for any measurement (Appendices A-

C). Multivariate analyses also indicated no overall frame 41 size effect. Interaction of frame sizes of steer with age of steers also was not statistically significant. Because the only significant effect suggested by analyses of variance was that due to age and not to frame size of steer, data for each frame size of steer were combined for presentation of results.

1. Fatty acid synthesis during growth

To study the importance of lactate as a precursor for fatty acids during growth of cattle, fatty acid synthesis from lactate and acetate was compared in adipose tissue taken from three major fat depots of growing cattle. Rates of lipogene- sis, expressed on a cellular basis, in subcutaneous, inter­ muscular and intramuscular adipose tissues, are shown in Tables

1-3, respectively. Fatty acid synthetic rates from lactate and acetate increased from 11 to 13 months of age in subcutaneous and

and intermuscular adipose tissues (Tables 1 and 2). Synthetic

rates from lactate in subcutaneous and intermuscular adipose

tissues increased significantly from 11 to 17 months of age; rates did not change significantly from 17 to 19 months of age.

Synthetic rates from acetate in these two tissues did not

change during growth from 13 to 19 months of age. Simple correlation coefficients for fatty acid synthetic rates from

lactate and acetate in subcutaneous and intermuscular adipose tissues were 0.37 (P <0.05; Appendix D) and 0.36 (P <0.05; Appendix E), respectively. 42

Table 1. Fatty acid synthesis from lactate and acetate in subcutaneous adipose tissue of steers during growth

Substrate Lactate Acetate (months) (nmoles substrate converted to fatty acid/ 2hr X 10® cells))

11 1051 ± 331^'^^ 2824 ± 1111^ 13 3685 ± 713b,d 9017 ± 2827^ 17 6771 ± 2174C 7604 ± 1268^ 19 9011 ± 1322° 6694 ± 894^

^Means ± SEM for eight steers ^'^Means in same column with different superscripts are significantly different (P <0.05). '^Significantly different (P <0.05) from that for acetate in same age group

Table 2. Fatty acid synthesis from lactate and acetate in intermuscular adipose tissue of steers during growth Substrate Age Lactate Acetate (months) (nmoles substrate converted to fatty acid/ 2hr X 10® cells)) 11 417 ± 113^'^'® 1271 ± 386° 13 1696 ± 378^'® 3192 ± 933d 17 3587 ± 1007^ 2387 ± 537^ 19 3268 ± 373^'^ 2939 ± 271^ fd

^Means ± SEM for eight steers unless noted otherwise

^Mean ± SEM for seven animals c d ' Means in same column with different superscripts are significantly different (P <0.050.

^Significantly different (P <0.05) from that for acetate in same age group 43

Rates of fatty acid synthesis from both lactate and ace­ tate increased with increasing age in intramuscular adipose tissue (Table 3). Synthetic rates from lactate increased about thirty-fivefold over the entire growing and fattening period, while synthetic rates from acetate increased only about four­ fold. Fatty acid synthetic rates were significantly greater from lactate than from acetate after 13 months of age. A simple correlation coefficient of 0.59 (P < 0.05; Appendix F) was observed for rates of fatty acid synthesis from lactate and acetate.

Table 3. Fatty acid synthesis from lactate and acetate in intramuscular adipose tissue of steers during growth Substrate Age Lactate Acetate (months) (nmoles substrate converted to fatty acid/ 2hr X 106 cells))

11 43 ± 11^'^ 185 ± 56^ 13 244 ± 41^ 297 ± 48^ 17 1491 ± 271^'^^'® 370 ± 133b /C 19 1448 ± 272^'® 722 ± 95^

^Mean ± SEM for eight steers unless noted otherwise

^Mean ± SEM for seven steers

c' d Means in same column with different superscripts are significantly different (P <0.05)

^Significantly different (P <0.05) from that for acetate in same age group 44

2. Activity of lipogenic enzymes Specific activities, hereafter simply called activities, of acetyl-CoA carboxylase, ATP-citrate lyase and selected

NADPH-generating enzymes during growth were determined to re­ late lipogenic capacity to possible rate-limiting enzyme activ­ ities. Another major objective was to determine if sufficient ATP-citrate lyase activity existed to support fatty acid syn­ thesis from lactate via the citrate cleavage pathway. Activity of the lipogenic enzymes for the three adipose tissue depots is shown in Tables 4 to 6.

Activity of acetyl-CoA carboxylase did not change signifi­ cantly during the growing period in any of the adipose tissue depots studied. In subcutaneous adipose tissue, activity of

ATP-citrate lyase increased from 11 to 13 months of age and did not change significantly thereafter. In intermuscular adipose tissue, ATP-citrate lyase activity increased during growth from 11 to 13 months of age and decreased from 13 to 19 months of age. Activity of ATP-citrate lyase did not increase until 19 months of age in intramuscular adipose tissues. Ac­ tivities of acetyl-CoA carboxylase and ATP-citrate lyase in intramuscular adipose tissue were greater than the same activ­ ities in subcutaneous and intermuscular adipose tissues", taken from cattle at all ages.

Activity of the principal NADPH-generating enzymes in­ creased in subcutaneous and intermuscular adipose tissues from Table 4. Activity of lipogenic enzymes and NADPH-generating enzymes in subcutaneous adipose tissue of steers during growth

Enzyme

Acetyl-CoA ATP-citrate Glucose-6- 6-Phospho- NADP- Age carboxylase lyase phosphate gluconate isocitrate dehydrogenase dehydrogenase dehydrogenase

(months) (nmoles substrate converted to product/(min x 10 cells))

a,b 11 17 ± 6 58 ± 13 5415 ± 1072 2277 ± 180 5337 ± 957

13 35 ± 6 310 ± 43 33503 ± 6120 11848 ± 3408 16300 ± 2097

17 30 ± 6 230 ± 74 27340 ± 5909 10231 ± 2194 19440 ± 3447

19 29 ± 6 216 ± 37 22502 ± 3564 9321 ± 1334 18010 ± 1626

^eans ± SEM for eight animals. b c ' Means with different superscripts in same column are significantly different (P <0.05). Table 5. Activity of lipogenic enzymes and NADPH-generating enzymes in intermuscular adipose tissue of steers during growth

Enzyme

Acetyl-CoA ATP-citrate Glucose-6- 6-Phospho- NADP- Age carboxylase lyase phosphate gluconate isocitrate dehydrogenase dehydrogenase dehydrogenase

(months) (nmoles substrate converted to product/(min x 10^ cells))

11 16 + ga.c 52 ± 11^ 5556 ± 1146^ 2371 ± 183^ 6625 537^

13 35 + ii" 230 ± 55"^ 17075 ± 2823^ 6629 ± 1444^ 11806 + 1050^

17 7 + 109 ± 33^ 15032 ± 3590^ 4502 ± 705^ 14270 ± 1770^

19 34 + lyb.d 96 + 21^'C 13942 ± 2274^'^ 5597 ± 797^'^ 16866 ± 2956^'^

^eans ± SEM for eight animals unless noted otherwise.

Cleans ± SEM for seven animals.

^'^eans in same column with different superscripts are significantly different (P <0.05). Table 6. Activity of lipogenic enzymes and NADPH-generating enzymes in intramuscular adipose tissue of steers during growth

Enzyme

Acetyl-CoA ATP-citrate Glucose-6- 6-Phospho- NADP- Age carboxylase lyase phosphate gluconate isocitrate dehydrogenase dehydrogenase dehydrogenase

(months) (nmoles substrate converted to product/(min x 10^ cells))

11 60 ± 26*'C 424 ± 50^ 3597 ± 811^ 1632 ± 273^ 3625 ± 546^

13 53 ± 13^ 449 ± 11^ 6730 ± 1507^ 3691 ± 785^ 3514 ± 371^

17 35 ± 9^'^ 440 ± 54^'^ 7705 ± 1594^'^ 2629 ± 458^'^ 6730 ± 1077^

19 48 ± 26^ 835 ± 146^ 12439 ± 2664® 3556 ± 400^ 7940 ± 881^

^eans ± SEM for eight animals unless noted otherwise.

^Means ± SEM for seven animals.

OjdjGjj^^ans in same column with different superscripts are significantly different (P <0.05). 48

11 to 13 months of age and did not change significantly during the remainder of the growing period (Tables 4 and 5).

In intramuscular adipose tissue, activity of glucose-6-phos- phate dehydrogenase increased significantly from 11 to 13 and

17 months of age and increased further by 19 months of age

(Table 6). Activity of 6-phosphogluconate dehydrogenase in­ creased from 11 to 13 months of age in intramuscular adipose

tissue and did not change thereafter. Activity of NADP-iso-

citrate dehydrogenase in intramuscular adipose tissue did not

change from 11 to 13 months of age but increased between 13 and

17 months of age and remained unchanged thereafter. At each

age and in each adipose tissue depot, activity of glucose-6-

phosphate dehydrogenase was an average of threefold greater

than that for 6-phosphogluconate dehydrogenase. Activities of

the three NADPH-generating enzymes in intramuscular adipose

tissue were lower than those observed in subcutaneous and

intermuscular adipose tissues at each respective age.

D. Discussion Pothoven and Beitz (1973) observed decreases in rates of

fatty acid synthesis in bovine adipose tissue with increasing

steer weight when rates were expressed on a tissue-weight basis.

Similar observations have been made for growing lambs (Ingle et

al., 1972) and rats (Benjamin et al., 1961). When rates were

expressed on a cellular basis, however, no decrease in fatty

acid synthesis was observed in rat adipose tissue with advancing 49 age (Greenwood et al., 1970). In the present study, fatty acid synthetic rates in bovine adipose tissues were expressed on a cellular basis. Whether lactate or acetate was utilized as the substrate, fatty acid synthesis rates were observed to increase with advancing age in the three major adipose tissue depots of growing and fattening cattle. It is evident that the capacity to synthesize fatty acids does not decrease with advancing age in cattle when data are expressed on a cellular basis.

When acetate was the substrate, previous studies have shown that rates of fatty acid synthesis are greater in subcu­ taneous adipose tissue than in internal or intramuscular adi­

pose tissues (Hood and Allen, 1975b; Pothoven et al., 1975;

Chakrabarty and Romans, 1972). In this study, fatty acid synthesis rates from lactate and acetate were greater at all

ages in subcutaneous than in intermuscular or in intramuscular

adipose tissues. This result further confirms the important

role of subcutaneous adipose tissue in lipid accretion in cattle.

As was found for fatty acid synthetic capacity, lipogenic

enzyme activities also tended to increase with increasing age

in all three adipose tissue depots. The notable exception,

however, was that acetyl-CoA carboxylase was not significantly

changed over the entire period from 11 to 19 months of age.

Similarly, little change in acetyl-CoA carboxylase was observed 50

in adipose tissue of growing swine by Anderson and Kaufman

(1973). No previous information is available on activities of

acetyl-CoA carboxylase of adipose tissue of growing cattle.

Increases in activity of ATP-citrate lyase and NADPH-generating

enzymes have been observed in growing swine (Hood and Allen,

1973a; Lee and Kaufman, 1974a). Pothoven and Beitz (1973) ob­

served slight, but not statistically significant, increases

in activity of glucose-6-phosphate dehydrogenase in bovine adi­

pose tissues with increasing animal weight; however, their data

were not expressed on a cellular basis. Hanson and Ballard

(1968) demonstrated that liver of mature ruminants had lower

specific activity of ATP-citrate lyase than liver of fetal

ruminants, but little information is available concerning

changes in activity of ATP-citrate lyase during post-weaning

growth in cattle.

Fatty acid synthetic rates and activities of key lipo-

genic enzymes in adipose tissues taken from the two frame sizes

of steers were not significantly different. In a recent report,

Scott and Prior (1980) also found no difference in activities

of acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase

and 6-phosphogluconate dehydrogenase in subcutaneous adipose tissue from small and large "frame" cattle.

Scott and Prior (1980) reported greater activity of fatty acid synthesis in subcutaneous adipose tissue of small frame

cattle compared to large frame cattle and greater activity of 51

NADP-isocitrate dehydrogenase in subcutaneous adipose tissue of large frame cattle compared to small frame cattle. The finding of no significant differences in lipogenic parameters of the two frame sizes of cattle is consistent with similari­ ties in observed carcass composition and adipose tissue eel- lularity of the two types of cattle used in the present study

(Cianzio, 1980).

One objective of the present study was to determine if sufficient ATP-citrate lyase activity existed to support lac­ tate conversion to fatty acids and to identify possible rate- limiting enzymes for conversion to fatty acids. Approximately sixfold greater activity of ATP-citrate lyase was observed in subcutaneous and intermuscular adipose tissues than is needed to support observed rates of fatty acid synthesis from lactate

(Table 7). Approximately sixtyfold more ATP-citrate lyase activity was observed in intramuscular adipose tissue than is needed for observed rates of fatty acid synthesis from lactate.

These results do suggest that fatty acid synthesis from lactate could occur via the citrate cleavage pathway in bovine adipose tissues,

Activities of major NADPH-generating enzymes in ruminants

(Baldwin et al., 1976) were considerably greater than those needed to supply the NADPH required for lipogenesis from lac­ tate in all three adipose tissue depots (Table 7). This result suggests that NADPH-generating capacity does not limit 52

Table 7. Relationship of enzymatic activities and fatty acid synthesis from lactate in bovine adipose tissues

Adipose tissue

Item Subcutaneous Intermuscular Intramuscular

(nmoles substrate; converted (or NADPH formed)/ (2hr X 10^ cells))

Lactate conversion to fatty acids 5130^ 2209 784

ATP-citrate (required) 5130 2209 784 lyase (observed) 24442^ 14857^ 64656^

NADPH (required) 10260 4408 1568 (observed) 5457681°'^ 3519292^ 1938759^

Acetyl-CoA (required) 5130 2209 784 carboxylase (observed) 3324 2750 6085^

^eans over all age groups.

^Represents sum of NADPH generation from glucose-6-phosphate dehydro­ genase, 6-phosphogluconate dehydrogenase and NADP-lsocitrate dehydrogenase.

"^Observed value is significantly different from required (P <0.05). Student's t-test was used to determine difference in values. 53 lipogenesis from lactate in bovine adipose tissue.

In subcutaneous and intermuscular adipose tissues, activi­ ties of acetyl-CoA carboxylase and rates of fatty acid synthe­ sis from lactate were not significantly different, which sug­ gests that activity of acetyl-CoA carboxylase limits the rate of fatty acid synthesis from lactate in these tissues. This result is consistent with the generally accepted rate-limiting role of acetyl-CoA carboxylase in fatty acid biosynthesis

(Lane et al., 1979). In a recent report. Smith and Prior (1980) also have suggested that acetyl-CoA carboxylase limits rate of fatty acid synthesis from lactate in subcutaneous bovine adipose tissue. Greater activity of acetyl-CoA car­ boxylase was observed in intramuscular adipose tissue than is needed to support fatty acid synthesis from lactate, which suggests that some other enzyme limits lipogenesis from lac­ tate in this adipose tissue.

Current concepts of lipogenesis in bovine adipose tissue state that glucose use is limited for fatty acid synthesis because of low citrate cleavage pathway activity (Ballard and

Hanson, 1967a; Bauman, 1976). On the basis of known metabolic pathways, lactate as well as glucose is catabolized to pyruvate as a common metabolic intermediate. Lactate as well a.o pyru­ vate is converted to fatty acids at rates several fold greater than glucose in bovine adipose tissue (Whitehurst et al., 1978).

Concentrations of specific isozymes of 54

(EC 1.1.1.27) suggest that lactate would be converted to pyruvate (Prior and Jacobson, 1979a). Furthermore, both pyru­ vate and lactate are decarboxylated before being converted to fatty acids (Prior and Jacobson, 1979b). Heretofore, the in­ volvement of the citrate cleavage pathway for lactate con­ version to fatty acids in bovine adipose tissue has not been demonstrated. Data in the present study clearly indicate that

ATP-citrate lyase activity was more than sufficient to support fatty acid synthesis from lactate in subcutaneous, intermuscu­ lar and intramuscular adipose tissues of cattle. Therefore, the citrate cleavage pathway, as described by Ballard and

Hanson (1967a) , seems operative in bovine adipose tissue and would explain the generation of cytosolic acetyl-CoA from lactate for fatty acid biosynthesis. 55

IV. METABOLISM OF PROPIONATE, GLUTAflATE, ACETATE, LACTATE AND GLUCOSE IN RUMINANT ADIPOSE TISSUES

A. Introduction

Acetate is the principal precursor for lipogenesis in adi­ pose tissue of ruminants (Ballard and Hanson, 1967a; Hood et al., 1972, Ingle et al., 1972). Low activity of the citrate cleavage pathway was proposed by Ballard and Hanson (1967a) to account for the low capacity of ruminants to use glucose for fatty acid synthesis. Subsequent studies have demonstrated that both lactate and pyruvate are fatty acid precursors in ruminants (Whitehurst et al., 1978; Prior, 1978; Prior and

Jacobson, 1979a, b). The use of pyruvate and lactate for fatty acid synthesis suggests that metabolic steps not involved in the citrate cleavage pathway might limit glucose conversion to fatty acids in ruminant adipose tissues.

The overall goal of the present study was to test the proposal that low citrate cleavage pathway activity does not limit glucose conversion to fatty acids in ruminant adipose tissues. One objective was to measure rates of 5- 14 C-gluta- mate, propionate and lactate conversions to fatty acids because the only known mechanisms for these substrates to be utilized for fatty acid synthesis is via the citrate cleavage pathway (Leveille and Hanson, 1966a, b). An additional ob­ jective was to determine the proportion of absorbed glucose catabolized via the pentose phosphate pathway to examine whether 56 glycolytic activity limits glucose conversion to fatty acids in ruminant adipose tissue.

B. Materials and Methods

1. Tissue sampling

Bovine subcutaneous and ovine perirenal adipose tissues were removed within 45 minutes after stunning and exsanguina- tion and kept at 37°C in a 0.9% NaCl solution buffered with 20 mM potassium phosphate, pH 7.4, until assayed. Backfat was

removed from over the 9th through 13th rib section of three

beef steers ranging in weight from 703 kg to 746 kg. Perirenal

adipose tissue was removed from three lambs ranging in weight

from 47 kg to 52 kg. Cattle and lambs were fed ad libitum a

high energy corn-based diet. Epididymal adipose tissue was

removed from male rats of the Charles River strain after de­ capitation. Rats weighed between 318 g and 370 g and were ad

libitum fed a 4% fat stock diet (Teklad Mouse/Rat Diet, ARS

Sprague Dawley Division of Mogul., Corp., Winfield, Iowa.

2. Assay of CO^, fatty acid and glycerol synthesis

Sections of adipose tissue weighing approximately 150 mg were incubated in triplicate at 37°C for 2 hours in 2+ 3 ml of Krebs-Ringer bicarbonate buffer, pH 7.4 (Ca -free;

Laser, 1961) that contained 0.3 lU of bovine insulin (Sigma

Chemical Co., St. Louis, Missouri). Radioactive substrates added to separate incubations were [1-^^C]-acetate, 57

D-[1-^'^C]-glucose (New England Nuclear Corp., Boston, 14 Massachusetts), D-[6- C]-glucose (New England Nuclear Corp., 14 Boston, Massachusetts), D-[6- C]-glucose (New England Nuclear Corp., Boston, Massachusetts), DL--glutamate (New

England Nuclear Corp., Boston, Massachusetts), L-[U-^^C]- lactate (New England Nuclear Corp., Boston, Massachusetts) and

[2-^'^C]-propionate (ICN Chemical and Radiosotopy Div., Irvine,

California. Incubations were conducted in siliconized flasks that were sealed with rubber septa; contents were gassed with

95% Og: 5% CO^ for 15 seconds.

Carbon dioxide released from acidified media and collected on filter paper wetted with 0.1 ml 30% NaOH was assayed for radioactivity as previously described (Pothoven and Beitz,

1973) with a Beckman LS-8000 liquid scintillation spectropho­ tometer. Quenching was corrected by the external standards method.

After collection of CO2'tissue sections were separated from incubation media rinsed with a 0.9% NaCl solution, placed in 20 ml of 2:1 (v/v) chloroform-methanol and shaken overnight on a wrist-action shaker. The resulting lipid extracts were washed as described by Folch et al. (1957) , evaporated and saponified for 2 hours at 85°C in 3% ethanolic NaOH. After re­ moval of nonsaponifiable lipids, fatty acids were extracted with hexanes (mixture of isomers of hexane) as described by

Pothoven and Beitz (1973). Hexanes were evaporated with 58 moving air, and residue was dissolved in scintillation solution for determination of radioactivity in fatty acids. The aqueous extract containing the glycerol was evaporated, and radio­ activity was determined by liquid scintillation counting.

3. Contribution of pentose phosphate pathway The percentage of glucose metabolized by the pentose phos­ phate pathway was calculated from glucose uptake and specific yields of products as described by Katz et al. (1966) and Katz and Wood (1963). Glucose concentration in incubation

media before and after incubation of adipose tissues was mea­

sured by glucose oxidase method with a commercial kit (Worth-

ington Biochemical Corp., Freehold, New Jersey). Glucose up­ take was calculated by difference. Specific yields of radio­

active products were measured as discussed by Katz and Wood

(1963). A specific yield of product is defined as the ratio

of isotope isolated in a product to total uptake of radioac­

tive substrate. Total uptake of isotope was considered to be

the uptake of substrate multiplied by its specific radioactiv­ ity (DPM/ymole).

4. Statistical analysis

Pairwise comparisons of treatment means were made with student's t-test (Snedecor and Cochran, 1967). Standard errors

for the difference of two means were computed from average of

individual treatment variances. 59

C. Results

1. Propionate, glutamate, lactate and acetate metabolism in bovine adipose~tissue

To evaluate the involvement of the citrate cleavage path­ way for fatty acid synthesis in ruminants, rates of propionate, glutamate, lactate and acetate conversions to fatty acids, glycerol and COg in bovine adipose tissue were determined and are shown in Table 8. Synthetic rates for fatty acids and glycerol from propionate were lowest of all substrates assayed and showed no concentration dependence. Rates of oxidation of propionate to COg also did not show a concentration dependence and at a concentration of 10 mM were lower (P <0.10) than oxidation rates of other substrates assayed at 10 mM concen­ tration. The low rates of propionate conversion to fatty acids are similar to those observed for cattle by Hood et al. (1972); greater rates of propionate oxidation of propionate to COg,

however, were observed by Hood and coworkers.

In contrast to those of propionate, rates of fatty acid synthesis from glutamate as well as the rates of oxidation of

glutamate to CO^ showed a concentration dependence. Calculation of the rate of glycerol synthesis from glutamate did not take

into account the loss of because of randomization after

formation of succinate in the tricarboxylic acid cycle. In­ corporation of from [5-^^C]-glutamate into fatty acids

represents a backward reaction of the tricarboxylic acid cycle 60

(glutamate ->• a-ketoglutarate ->• isocitrate citrate acetyl-

CoA + oxaloacetate; Madsen et al., 1964a, b; Amedo and Haft,

1965) and, because of the asymmetric behavior of citrate in

the reaction catalyzed by ATP-citrate lyase (Bhaduri and

Srere, 1963; Spencer and Lowenstein, 1962), indirectly measures

activity of ATP-citrate lyase necessary for fatty acid synthe­

sis from glutamate. Rates of fatty acid synthesis from gluta­

mate tend to be lower than those for lactate, but greater than

those for propionate at similar concentrations. These precur­

sor-product relationships suggest that lactate v/as converted

to fatty acids by way of the citrate cleavage pathway.

Table 8 provides additional support for the major impor­ tance of acetate as a fatty acid precursor in ruminants. Only

at a concentration of 100 mM did rates of fatty acid synthesis

from lactate approach or exceed those observed from 25 mM acetate. At a concentration of 1 mM, rates of fatty acid syn­

thesis from acetate were much greater than from the other sub­ strates tested at similar concentrations.

2. Lactate, acetate and glucose metabolism in ovine adipose tissue

Rates of lactate, acetate and glucose conversions to

fatty acids, glycerol and CO2 by perirenal adipose tissue from

lambs are shown in Table 9. Fatty acid synthetic rates from

lactate and acetate were similar and approximately 5-fold

greater than that from glucose. Lactate and glucose were used 61

at similar rates for glycerol synthesis. Low rates of glycerol synthesis from acetate represents recycling of radioactive

label and thus not net synthesis of glycerol. Oxidation of

lactate, acetate and glucose to CO^ occurred at a similar rate.

3. Activity of pentose phosphate pathway

To obtain further insight into alternative explanations

to that of low activity of citrate cleavage pathway proposed

by Ballard and Hanson (1967a) for poor ability of ruminants

to use glucose for fatty acid synthesis, the percentage of glucose catabolism via the pentose phosphate pathway was deter­

mined for subcutaneous adipose tissue from cattle and peri­

renal adipose tissue from lambs. Studies also were conducted

with rat adipose tissue to compare with previous investigations with nonruminants (Katz et al., 1966; Landau and Katz, 1964).

Measurements were made with different cattle from those cited

in Table 8, but were made with same lambs used for experiments

of Table 9. Rates of fatty acid synthesis from lactate (10 mM

+ 5 mM glucose), acetate (10 mM + 5 mM glucose) and glucose

(5 mlM) in adipose tissue of cattle used in these experiments

were 871 + 91, 1434 + 90 and 260 + 71 nmole substrate converted

to fatty acids per gram tissue per 2 hours, respectively. Pentose phosphate pathway activity was estimated by deter­ mining the metabolic rate of [1-^^C]-glucose and [6-^^C]-

glucose as described by Katz et al. (1966). Specific yields 14 of CO2 from [1- C]-glucose were greater than those from 62

Table 8. Metabolism of propionate, glutamate, lactate and acetate by subcutaneous adipose tissue in cattle

Substrate Cone. Rate of synthesis Fatty acids Glycerol COg (nmole substrate converted/(2hr x g tissue))

Propionate 1 9 + 2^ .3 + .2 136 75

10 12 + 5 1 ± .3 127 ± 27

L-Glutamate 1 42 ± 6 10 ± 5 103 ± 6

10 125 + 59 4 + 1 238 + 55^

L-Lactate 1 93 + 57 5 + 3 202 100

10 327 + 121 32 ± 15 267 ± 17

100 1316 ± 176^ 97 + 50 1165 752^

Acetate ^ 1 859 + 140 2. 6 ± .1 207 + 55

25 1082 + 294 3.4 ± .7 1823 252°

^Means ± SEM for three steers.

^Incubation media also contained 5 mM glucose.

^Means in a column and from same substrate are signifi­ cantly different (P <0.1).

[6-^'^C]-glucose in both bovine and ovine adipose tissues

(Table 10). Greater yields of CO2 from [1-^^C]-glucose than 14 from [6- C]-glucose also have been observed in epididvmal adi­ pose tissue of rats (Katz et al., 1966; Winegrad and Reynold,

1958), in abdominal adipose tissue of nonlactating cows

(Baldwin et al., 1973) and in liver, kidney and brain of sheep 63

Table 9. Metabolism of lactate, acetate and glucose by perirenal adipose tissue of lambs

Substrate Rate of synthesis

Fatty acids Glycerol COg (nmole substrate converted/(2hr x g tissue))

Lactate^ 306 ± 51^'^ 14 ± 2^ 240 ± 46^ Acetate^ 343 ± 108^ 3 ± 1® 166 ± 36^'^

Glucose^ 62 ± 22^ 12 ± 3^ 107 ± 39®

^Incubation media contained 10 mM lactate or acetate in presence of 5 mM glucose.

^Means ± SEM for three lambs.

"^Incubation media contained 5 mM glucose.

"^'^Means in same column with different superscript are significantly different (P <0.05).

(Raggi et al,, 1961). Specific yields of fatty acids from [1- 14 C]-glucose were greater than those from [6-^'^C]-glucose. Specific yields of fatty acid from both labeled were similar in bovine and ovine adipose tissues. Specific yields of glycerol from

[1-^^C]-glucose and [6-^^C-glucose were similar in bovine adipose tissue but lower from [1-^^C-glucose than from [6-^^C]- glucose than from [6-^'^C]-glucose in ovine adipose tissue. The specific yields of [^^C]-fatty acids and [^'^C]-glycerol are lower than those observed in rat adipose tissue (Katz et al., Table 10. Specific yields of CO2j fatty acids and glycerol from specifically labelled glucoses in subcutaneous bovine and perirenal ovine adipose tissues

Species Specific yield of product SL Glucose uptake y b

Position (nmole/(2hr x g of Fatty acid Glycerol tissue)) label

Cattle^ 1 .178 + .058 .053 + .014 .021 + .005 1.99 ± .40 .772 ± .109

6 .029 + .007 .077 + .029 .028 + .009

Sheep^ 1 .093 + .028 .050 + .016 .004 + .001^ 1.31 ± .15 .645 ± .025

6 .033 + .013^ .072 + .022 .014 + .003^

^Specific yield is defined in text.

^Ratio of specific yields from [l-^^C]-glucose to [6-^^C]-glucose.

Slean ± SEM for three steers weighing from 492 kg to 528 kg.

^ean ± SEM for three lambs.

^Value is significantly different than corresponding value for bovine adipose tissue (P <0.05).

^Value is significantly different from value for [1-^'^C]-glucose (P <0.10). 65

1966). Lactate production was not quantitated in the present study but was quantitated by Katz et al. (1966); differences in lactate synthesis could account for the lower specific yields in ruminant adipose tissue.

Rates of glucose uptake observed in subcutaneous adipose tissue of cattle tended to be greater (P >0.05) than those ob­ served in adipose tissue of lambs. Rates of glucose uptake are quite low compared with those of rat adipose tissue (Katz et al., 1966). This observation is consistent with the finding of low glucose phosphorylating activities in ruminant liver

(Ballard and Oliver, 1964; Ballard, 1965) and in ruminant adipose tissue (Raggi et al., 1963) compared with those in liver and adipose tissue of rats.

The ratio (Y) of [^^C] yields in fatty acids from [1-^^C]- 14 glucose and from [6- C]-glucose observed in bovine adipose tissue was similar to that observed in ovine adipose tissue.

The ratio was less than 1 in adipose tissue of both ruminant species and was similar to the ratio of 0.58 estimated for adipose tissue of nonlactating dairy cows (Baldwin et al.,

1973).

Using the data in Table 10, estimates of the percentage

of glucose catabolism occurring via the pentose phosphate

pathway in ruminant adipose tissue were calculated by method

^, B and C of Katz et al. (1966). Method A is based on

specific yields of CO2 from [1-^^C]-glucose and [6-^^C]-glucose 66 method B is based on the ratio of fatty acids synthesized from

[1-^'^'C]-glucose and [6-^'^C]-glucose and method C is a combina­

tion of methods A and B (Table 11). Percentage of glucose catabolism via the pentose phosphate pathway in rat adipose, as calculated by method B was 22%, which is similar to values observed by Katz et al. (1966) and Landau and Katz (1964) for same tissue. The average value of the three methods in bovine adipose tissue was 7.3%, and all three methods were in agree­ ment with each other. The average value for pentose phosphate metabolism in ovine adipose tissue was 6.8 percent. Method B gave a greater estimate of pentose cycle activity than methods

A and C for ovine adipose tissue.

D. Discussion

Both acetate and lactate were used for fatty acid synthe­ sis at greater rates than glucose in adipose tissues of cattle and lambs. Results of substrate use for fatty acid synthesis in ovine adipose tissue are similar to those of Whitehurst et al.

(1978) for bovine adipose tissue and suggested that the limita­ tion of glucose conversion to fatty acids in bovine and ovine

adipose tissues are similar. Numerous previous investigators

have observed low rates of glucose conversion to fatty acids

in ruminant adipose tissue and liver (Ballard and Hanson,

1967a; Ingle et al., 1972; Hood et al., 1972; Whitehurst et al.,

1978). In adipose tissues of sheep and cattle, low rates of

glucose conversion to fatty acids have been correlated with 67

Table 11. Contribution of pentose phosphate pathway to glucose metabolism in ruminant and nonruminant adipose tissue

Species Method

A B (% ± SEM)

Cattle 6.0 ± 2.2 9.8 ± 5.1 6.1 ± 2.2

Sheep 2.2 ± .6^ 15.6 ± 1.4 2.5 ± .7

Rat ND^ 22 ± 11 ND^

^Methods A, B and C of Katz et al. (1966). ^PC = S/(3-2S) X100%.

GI^Q = Specific yield of CO^ from [1-^^C]-glucose.

G6Ç,Q = Specific yield of CO2 from [6-^^C]-glucose.

PC = Percentage of glucose catabolism via the pentose phosphate pathway. ^PC = (1-Y)/(1+2Y) X 100%. Y = Ratio of specific yields of fatty acid from [1-^^C]- glucose to those from [6-^'^C]-glucose. ^PC = S^/(3-2S^).

^Value is significantly different from value for adipose tissue of cattle (P <0.10).

f Not determined. 68 comparatively low activity of the citrate cleavage pathway

(Ballard and Hanson, 1967). Lactate was observed to be a fatty acid precursor in ovine adipose tissue in the present study, which further confirms earlier work by Prior (1978) who demonstrated that lactate could be used as a fatty acid pre­ cursor in ovine adipose tissue.

In addition to lactate, pyruvate also has been observed to be converted to fatty acids at greater rates than glucose (Whitehurst et al., 1978). Prior and Jacobson (1979b) have shown that pyruvate and lactate are both decarboxylated before being converted to fatty acids by bovine adipose tissue. These observations of lactate and pyruvate metabolism by ruminant adipose tissues are difficult to reconcile with the commonly accepted explanation that low citrate cleavage pathway activity limits use of glucose for fatty acid synthesis in lipogenic tissues of ruminants.

In the present investigation, an indirect approach has been used to examine the hypothesis that low citrate cleavage activity does not limit glucose use for lipogenesis. Similar rates of fatty acid synthesis were observed for both glutamate and lactate, suggesting that ATP-citrate lyase activity is present to support lipogenesis from lactate. Glutamate la­ belled in the number 5 carbon can only be used for fatty acid synthesis via the citrate cleavage pathway (Leveille and Hanson, 1966a, b). 69

Propionate also should be converted to fatty acids via the citrate cleavage pathway; propionate, however, was used at com­ paratively low rates for lipogenesis in adipose tissue of cattle. Low rates of propionate use for fatty acid synthesis also has been demonstrated by Hood et al. (1972). This ob­ servation suggests that use of propionate for fatty acid syn­ thesis probably is limited at some reaction other than that catalyzed by ATP-citrate lyase. Propionate conversion to in­ termediates of the tricarboxylic acid cycle seem not to be rate limiting, because propionate was oxidized to CO2 at rates greater than that for fatty acid synthesis and similar to those of glutamate and lactate oxidation to COg.

To gain further insight into possible explanations for the inability of ruminant adipose tissue to use glucose for fatty acid synthesis, glucose catabolism via the pentose phosphate pathway was estimated. Catabolism of glucose via the pentose phosphate pathway in both bovine and ovine adipose tissues accounted for less than 8% of the glucose metabolized. This estimate is lower than that found for rat adipose tissue (Table

11 and Landau and Katz, 1964; Katz et al,, 1966). Studies with

[1-^^C]-glucose and [6-^^C]-glucose showed that less than 30% of the total glucose uptake could be accounted for by COg, fatty acids and glyceride-glycerol. Yang and Baldwin (1973) were able to account for only 50% of catabolized glucose in COg, fatty acids and glycerol in isolated bovine adipocytes; this 70

result suggests significant catabolism of glucose to other, as yet unidentified, metabolites occurs in ruminant adipose tissues. Clearly, much additional work is needed to quantitate the metabolic fates of all glucose in ruminant adipose tissue.

One of the more striking observations concerning glucose metabolism in bovine and ovine adipose tissues was the low rate of glucose uptake compared with rates reported for rat adipose tissue (Katz et al., 1956). This observation is con­ sistent with the finding of low glucokinase activity in bovine adipose tissue (Raggi et al., 1963) and suggests that low rates of glucose conversion to fatty acids observed in ruminant adipose tissue are because of low rates of glucose phosphoryla­ tion. Recently, Smith and Prior (1980) suggested that either hexokinase or pyruvate kinase might limit glucose conversion to fatty acids; however, the limiting step remains to be elucidated. 71

V. EXAMINATION OF A LOGNORMAL EQUATION TO DESCRIBE DISTRIBUTIONS OF DIAMETERS TO BOVINE ADIPOCYTES

A. Introduction

Determination of adipocyte size and number is necessary to understand growth and development of adipose tissue of meat an­ imals. One method for measuring adipose tissue cellularity in­ volves fixation of fat cells in adipose tissue slices with os­ mium tetroxide, followed by counting and sizing of fixed adi­ pocytes with an automatic particle counter (Hirsch and Gallian,

1968). The method has several advantages that include the ease and accuracy with which great numbers of cells can be counted and the accuracy with which the distribution of adipocytes can be determined. Sjostrom et al. (1971) suggested that adi­ pocyte size distributions were normally distributed; however, distributions of adipocytes presented by some workers are skewed (Hood and Allen, 1975a; Hood and Thornton, 1979).

Because skewed size distributions of adipocytes are observed, the size distribution of adipocytes may not be most accurately described as a normal or Gaussian distribution function. A possibility is that populations of adipocytes are lognormally distributed.

Therefore, the objective of this study was to examine the use of a lognormal distribution function to describe the size distributions of bovine adipocytes. A computational form of the lognormal distribution described by Siano (1969) and 72

Siano and Metzler (1969) was used to model size distributions of adipocytes determined with an automatic particle counter.

B. Materials and Methods

1. Adipose tissue sampling and fixation

Adipose tissue samples were collected from beef steers after stunning and exsanguination and kept in a 0.9% NaCl solution of 37°C until preparation for fixation. Steers ranged from 11 to 15 months of age, and all were fed ad libitum a ration consisting of 72% ground shelled corn, 22%

alfalfa-brome haylage or oatlage and 6% of a pelleted 32%

protein supplement. Subcutaneous adipose tissue was taken from either the 10th through 12th rib section or from the

brisket region. Intermuscular adipose tissue was separated

from between the iliocostalis thoracis and the longissimus

dorsi of the 10th through 12th rib section. Mesenteric

adipose tissue was collected from tissue between the caudal end of the cecum and the small intestine. Adipose tissue

samples from several anatomical sites were taken to test the

general applicability of the proposed method.

A weighed (approximately 200 mg) section of adipose tissue

was fixed with 2% osmium tetroxide in 50 mM collidine hydro­ chloride buffer solution at pH 7.6 as described by Hirsch and

Gallian (1968). Connective tissue debris was solubilized with 8 M urea as described by Etherton et al. (1977) , and adipocytes 73 were freed by washing with a 0.9% NaCl solution containing

0.01% Triton X-100 through a 250 ym nylon mesh filter and collected onto a 20 ym nylon mesh filter. Adipocytes were washed into a weighed 400 ml beaker with a 0.9% NaCl solu­ tion; volume was increased to 350 ml with the NaCl solution, and the beaker was reweighed.

2. Adipocyte counting

Distribution of adipocyte sizes was determined by count­ ing samples with a model ZB Coulter Counter, Coulter Elec­ tronics, Inc., Hialeah, Florida, in the expanded or loga­ rithmic mode. A 400 ym aperature was used, and a 90.5 ym standard of polystyrene beads was used to determine volume for each of the instrument's 100 channels. Standard parti­ cles and the fixed adipocytes were assumed to be spherical.

3. Analysis of particle counter data

Particle counter data were analyzed by fitting number of cells in each diameter class to a normal distribution function and a lognormal distribution function. The form of the normal distribution function used is shown by Equation 1, where f(t) is the number of cells in a diameter class, yg is the param­ eter adjusting the height of the distribution and t is defined by Equation 2.

f(t) = y^ (1/2TT)^ exp (-t^/2) (1) 74

t = (x-ij)/o (2)

The parameter y is the mean diameter of the population of

cells, and a is the population's standard deviation; x is the adipocyte's diameter. A nonlinear regression program was

utilized to fit particle counter data to Equation 1 (Barr et al., 1979).

A computer program described by Siano (1969) and Siano

and Metzler (1969) for the analysis of electronic absorption

spectra was utilized to fit particle counter data to a log- normal distribution function (Equation 3), where g(z) is

the number of cells of a particular diameter, yg is the

maximal number of cells, p is a skewness estimate (Equation

4), z is the diameter of the adipocyte, ZQ is the diameter at the maximal number of cells and w is the width of the curve

at half maximal number of cells (Equation 5).

g(z) = yg exp {-5,n (2)/5,n(p) [ £n{[ ( z-Zq)( p^-1)/wp] +1}]'} (3)

P = (Z2-Zq)/(ZQ-Z^) (4)

w = Zg-Z^ (5)

The parameters z^ and z^ are the diameters at the half maximal

number of cells of the left and right side of the curve, re­ spectively (Figure 3). Equation 3 is a modification of a form

of the lognormal frequency function described by Yaun (1933). Figure 3. Adipocyte diameter distribution of intermuscular adipose tissue of a13-month-old steer. Animal is number 347 (Table 12). Computational parameters are: yç, peak height; yo/2' half height; and Z2,

diameters at half height; and zq, diameter at peak height (see text). Solid line indicates predicted values from lognormal model. Points are observed values from automatic particle counter. 76

900

600

00 _J LU

300

170 270 DIAMETER (//m) 77

Calculation of mean, mode, median, standard deviation and higher moments are discussed by Yaun (1933).

Some modifications of the computer program of Siano (1969) and Siano and Metzler (1969) were made and included

increasing computational precision from single to double and

replacing subroutine FMCG of Scientific Subroutine Package,

International Business Machines Corp., White Plains, New York,

with subroutine ZXCGR of the International Mathematical and Statistical Library, International Mathematical and Statistical

Library, Brandenburg, Texas. Both subroutines perform function

minimization by the conjugate gradient technique, but the

latter subroutine allows specification of the initial step in

the minimization process, which can reduce computation time. Residual sum of squares was minimized by the computer program.

C. Results

Examples of modelling adipocyte size distributions ob­

tained with a particle counter to a lognormal distribution

function (Equation 3) are shown in Figures 3-6. Figures 3 and

4 represented samples of intermuscular adipose tissue from

two different steers who were 13 months of age. Figure 5 is

a sample of subcutaneous adipose tissue from a 15-month-old

steer whose adipocytes were of a single mode; Figure 6 was a

sample of subcutaneous adipose tissue of an 11-month old steer

in which the population of adipocytes was bimodal. Figure 4. Adipocyte diameter distribution from intermuscular adipose tissue of 13-month-old steer. Animal is number 242 (Table 12). Solid line indicates pre­ dicted values from lognormal model. Points are observed values from automatic particle counter. 79

900

600

(/)_j _j LU (_) O z:

300

170 270 DIAMETER (jjm) Figure 5. Adipocyte diameter distribution from subcutaneous adipose tissue (backfat) of 15-month-old steer. Animal is number 487 (Table 12). Solid line indi­ cates predicted values from lognormal model. Points are observed values from automatic particle counter. 81

900

600

300

69 169" 269

DIAMETER ifim) Figure 6. Adipocyte diameter distribution of subcutaneous adi­ pose tissue (brisket region) of 11-month-old steer. Animal is number 411 (Table 13). Solid line indicates predicted values from lognormal model. The upper line on peak at 37.5 ym diameter (Table 13) is pre­ dicted line for sum of two lognormal distributions and middle line is predicted for the peak itself. The lower line is the extrapolated predicted value for distribution with peak at 96.9 ym in diameter Table 13). Points are observed values from automatic particle counter. 83

720

480

00 _J LU

240

69 169 269 DIAMETER (// m) 84

Osmium-fixed adipocytes from the sample represented by

Figure 6 contained at lease two modes and possibly a third (Figure 7). Size distributions of adipocytes shown in Figures

3-6 displayed shoulders on the larger diameter side of the peak. Population parameters estimated by fitting a lognormal model to adipocyte size distributions are presented in Table

12. With three exceptions, modes and medians of adipocyte size distributions were larger than the means. The mode of a distribution is the location of the maximum of the distri­ bution function, and the value at above which or below which half of the distribution lies is referred to as the median

(Mood et al., 1974). For a normal distribution, the mean, mode and median are equal. The mean adipocyte diameter of all samples of subcutaneous adipose tissue was 102 ym, which is similar to the value of 106 ym observed by Hood and Allen

(1975a) for subcutaneous adipocytes of Holstein steers at

14 months of age. The normalized third and fourth moments are measures of skewness and peakedness (kurtosis); the values obtained for these parameters by fitting a lognormal model to size distri­ butions of adipocytes also are shown in Table 12. With three exceptions, the third and fourth moments were close to 0 and

3. Values of 0 and 3 are expected values of the normalized third and fourth moments of a normal distribution (Snedecor Figure 7. Adipocyte diameter distribution of subcutaneous adipose tissue (brisket region) of 11-month-old steer. Data were obtained by microscopic examination of osmium tetroxide-fixed cells, and sample was same as used with automatic particle counter as displayed in Figure 6. Microscopic sizing of adipocytes was performed with a Nikon Model M inverted microscope. PERCENT OF TOTAL

N) 00 en 4=- T

(D 1

00 ro 00 3 en m —I m I

O) jr-

ro ro kO Table 12. Parameters estimated by fitting lognormal distribution to automatic particle counter data for various adipose tissue locations in beef steers

Item Animal 543 535 527 487 347 242 214 127 080 060

Age (mo) 13 13 13 15 13 13 11 13 13 11 Carcass wt. (kg) 261 248 246 298 234 278 222 221 264 219 Location SQ^ ME^ SQ* SQ^ IN^ IN'^ ME^ ME^ SQ* IN^ Mean (ym) 90.1 112.6 113.4 120.7 118.7 114.6 106.2 127.5 81.9 107.0 Mode (ym) 93.9 125.5 106.4 125.6 126.7 117.5 106.4 127.0 101.5 104.8 Median (ym) 91.4 123.6 111.1 122.4 121.5 115.6 106.3 127.3 88.9 106.2 Third Moment .30 .10 .19 .26 .51 .10 .00 .01 1.59 .05 Fourth Moment 3.05 3.18 3.33 3.47 3.92 3.18 3.00 3.01 5.95 3.09 P® .93 .88 1.18 .82 .76 .88 .99 1.03 .64 1.09

^Subcutaneous adipose tissue (brisket region).

Mesenteric adipose tissue.

'^Subcutaneous adipose tissue (backfat) .

^Intermuscular adipose tissue.

^Parameter p is estimate of skewness at half height; p would equal 1.0 if skewness were absent. 88

and Cochran, 1967). The parameter p (Equation 4) also is an indicator of skewness and would be equal to 1 for a normal distribution

(Siano, 1969). Considerable variation was displayed in this parameter for estimating skewness of adipocyte size distri­ butions (Table 12). Values of p < 1 indicated negative skew­ ing and values of p > 1 indicated positive skewing.

Population parameters estimated for individual peaks from a bimodal population of adipocytes modelled with a lognormal distribution function are shown in Table 13. Means of the two

Table 13. Parameter estimates for bimodal adipocyte distri­ bution from subcutaneous adipose tissue (brisket region) of a beef steer at 11 months of age^'

Peak Item A B

Mean (ym) 34.7 96.9 Mode (ym) 37.5 115.2 Median (ym) 35.6 103.6 Std. Dev. 25.3 48.2 Third Moment .18 2.25 Fourth Moment 3. 32 7.26 P . 84 .59

The fitting procedure was to the sum of two lognormal distribution functions. Carcass weight of steer was 160 kg. 89 peaks were 34.7 ym and 96.9 ym and were smaller in both cases than their respective modes or medians. The standard deviation and normalized third moment for the peak with a mean of 34.7 ym (A) were smaller than the same parameters for the peak with a mean of 96.9 ym (B). The normalized fourth moment was smaller for peak B. Negative skewing was indicated for both peaks.

To compare normal and lognormal distribution functions for modelling size distributions of adipocytes, particle count­ ing data for samples indicated in Table 12 were fitted to Equa­ tion 1, and the results are shown in Table 14. In general, there was little difference in the mean adipocyte diameter estimated by either a normal or lognormal model of the adi­ pocyte populations. When data were modelled with normal dis­ tribution function, the standard deviation of the adipocyte populations was smaller.

To determine whether the normal or the lognormal model of adipocyte size distributions was better, chi square (x^) was calculated for each model and recorded in Table 14. Values of generally were greater for adipocyte size distributions modelled with a normal distribution; however, large values of

X^ were observed for both models.

The large values of can be attributed to specific regions of the populations of adipocytes as shown in Table 15, which shows the contribution to x^ for three regions of the adipocyte size distributions. On the smaller diameter side Table 14. Comparison of normal and lognormal models for describing populations of bovine adipocytes^

Item Distribution Animal 543 535 527 487 347 242 214 127 080 060

Mean (ym) Normal 92 124 109 123 123 116 106 127 93 106 Lognormal 90 123 113 121 119 115 106 128 82 107

Standard Normal 42 18 34 19 23 18 10 12 32 19 Deviation Lognormal 83 35 73 35 42 34 20 24 59 40 x'" Normal 102 783 152 430 838 347 1105 280 673 284

Lognormal 73 1180 65 139 200 170 1422 261 229 231

formal model refers to Equation 1 and lognormal model refers to Equation 3 in text.

was calculated as Z (Residual) ^ Predicted values <5 were omitted from calculation of • ^ Predicted Table 15. Contributions to for specific regions of adipocyte size distribution

Region Model Animal 543 535 527 487 347 242 214 127 080 060

Left Normal 11^ 536 10 360 730 296 365 65 132 2 Lognormal 12 118 16 33 49 49 380 96 13 161 Range of diameters (ym) <88 <88 <88 <88 <88 <88 <88 <97 <47 <88

Center Normal 22 84* 31 68* 83* 36* 35* 13 123* 88* Lognormal 22 117* 22 47* 34* 26* 43* 17 54* 30* Range of diameters (ym) 80-163 88-163 88-163 88-163 88-163 88-142 88-129 97-152 47-109 88-153

Right Normal 69 162 111 2 24 14 705 202 417 193 Lognormal 39 944 27 59 116 96 999 148 161 39 Range of diameters (ym) >163 >163 >163 >163 >163 >142 >129 >152 >109 >153

Values are E for specific region.

^Values of are larger than those expected for degrees of freedom in center region (p <0.05). 92 of size distributions (left) and the larger diameter side

(right), the contribution to generally was larger than that observed in the center of the adipocyte size distribution.

On the left side of the adipocyte distributions, no general trend for either model to have smaller contributions to was observed; on the right side of the adipocyte distribu­ tions, the lognormal model generally had larger contributions to x^- In the center region of the size distribution, the lognormal model was observed to provide a better fit to the adipocyte size distribution as evidenced by the usually smaller contribution to x^«

D. Discussion

Lognormal distributions have been used to describe the size of particles from a crumbling cookie (Koch, 1966), ultra­ violet spectra of molecules (Siano and Metzler, 1969), fre­ quency distributions of colloidal particle sizes (Herdan,

1953) and the distribution of financial incomes (Aitchison and Brown, 1957). Koch (1969) has suggested that size dis­ tributions of bacterial cells approach lognormality. Many diverse phenomena apparently can be described by a lognormal distribution.

In previous studies of the distribution of adipocyte sizes, Sjostrom et al. (1971) concluded that distribution of adipocytes from rat adipose tissue determined by counting osmium tetroxide-fixed adipocytes with an automatic particle 93 counter were normally distributed. Mersman et al. (197 3) modelled adipocyte diameter distributions from swine adipose tissues with a Gaussian distribution function and reported observing little skewness of the distribution of adipocyte sizes. Hood and Allen (1975a) and Hood and Thornton (1979), however, have presented frequency distributions of adipocytes from bovine and ovine adipose tissues, respectively, that are skewed. Because varying degrees of skewness are observed for distributions of adipocyte sizes, some distribution function other than a normal distribution function may more adequately describe the distribution of adipocyte sizes.

In the present investigations, a lognormal distribution function was used to describe the distribution of bovine adi­ pocytes. Seven of the ten samples examined displayed only slight departures from normality as evidenced by the third and fourth moments being close to 0 and 3, respectively.

The parameter p, which also measures skewness, was always less than 1 for the seven distributions that were close to normal­ ity. Three size distributions of adipocytes showed consider­ ably more departure from normality as evidenced by third and fourth moments greater than 0 and 3, respectively; these same populations had skewness estimates (p) greater than 1, indic­ ative of positive skewness. These results suggested that any distribution function chosen to describe size distributions of adipocytes should be capable of dealing with varying 94 degrees of skewness. The method presented in this study is capable of dealing with varying degrees of skewness observed. Comparison of normal and lognormal models for describing size distributions of adipocytes suggested that a lognormal model was more appropriate for describing size distributions of bovine adipocytes. Lower values of generally were ob­ served for size distributions modelled with a lognormal dis­ tribution function. Both models of adipocyte size distribu­ tion displayed weaker fits to the data in the left and right extremes of the populations. A greater standard deviation of the size distribution was estimated by a lognormal model, but both models estimated similar means for the size distribution.

In summary, a lognormal distribution function seems to more accurately describe size distributions of adipocytes from beef steers than other tested distribution functions. The lognormal distribution function described by Siano (1969) and Siano and Metzler (1969) provides a computationally convenient method for describing size distributions of adi­ pocytes and obtaining estimates of population parameters such as the mean, mode, median, standard deviation, normal­ ized third and fourth modents and p, an estimator of skewness.

An advantage of the method is that data collected with a particle counter can be used directly to obtain the population parameters of interest. 95

VI. DISCUSSION AND SUMMARY

Adipose tissue increases in mass by either cellular hypertrophy or hyperplasia or both. The studies presented in the preceeding sections have dealt with the development of adipose tissue of ruminants. The specific aspects of adipose tissue development investigated were:

1) The changes in adipose tissue lipogenic capacity and enzymatic activities during growth of cattle,

2) The role of the citrate cleavage pathway for gener­ ation of cytosolic acetyl-CoA in the biosynthesis of fatty acids in ruminant adipose tissue,

3) The limitation in the use of glucose as a fatty acid precursor in ruminant adipose tissues and

4) The distribution of adipocyte sizes in adipose of cattle.

The remaining discussion will be divided according to the aforementioned topics.

A. Changes in Fatty Acid Synthesis and Lipogenic Enzymes during Growth Pothoven and Beitz (1973) observed that rates of fatty acid synthesis in adipose tissue of Holstein steers, expressed on a tissue-weight basis, decreased with increasing animal weight. Expressed on a tissue-weight basis, rates of fatty acid synthesis also have been observed to decrease with ad­ vancing age in adipose tissue of lambs (Ingle et al., 1972) 96

and in adipose tissue of rats (Benjamin et al., 1961). In aging rats, however, no decrease in rates of fatty acid synthe­ sis was observed when data were expressed on a cellular basis (Greenwood et al., 1970). In the present study, rates of fatty acid synthesis, expressed on a cellular basis, increased with advancing age in subcutaneous, intermuscular and intra­ muscular adipose tissues of growing cattle when either lactate or acetate was used as the precursor. This observation sug­ gests that ability to synthesize fatty acids does not de­ crease with advancing age in cattle.

Activities of ATP-citrate lyase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-isoci- trate dehydrogenase, expressed on a cellular basis, also in­ creased with advancing age. Increases in activities of ATP- citrate lyase, glucose-6-phosphate dehydrogenase, 6-phospho­ gluconate dehydrogenase have been observed in adipose tissues of growing swine (Hood and Allen, 1975a; Lee and Kaufman,

1974b). Pothoven and Beitz (1973) observed slight increases in specific activity, expressed on a per mg protein basis, of glucose-6-phosphate dehydrogenase in subcutaneous , perirenal and omental adipose tissues of cattle with increasing body weight. No previous; information concerning changes in activi­ ties of ATP-citrate lyase and 6-phosphogluconate and NADP- isocitrate dehydrogenases during post-weaning growth of cattle is available. 97

Activity of acetyl-CoA carboxylase in subcutaneous, inter­ muscular and intramuscular adipose tissues of cattle did not change significantly during growth from 11 to 19 months of age. Anderson and Kaufman (1973) have observed little change in activity of acetyl-CoA carboxylase in subcutaneous adipose tissue of growing swine. No previous information concerning activity of acetyl-CoA carboxylase in growing cattle is avail­ able.

One of the objectives of the present study was to compare fatty acid synthetic capacity and lipogenic enzyme activities in crossbred cattle of two frame sizes that were based pri­ marily according to body weight of cattle at 180 days of age.

Statistically significant differences in the measured proper­ ties between the steers of the two frame sizes, however, were not observed. Scott and Prior (1980) found greater activity of fatty acid synthetase in subcutaneous adipose tissue of small frame cattle (Angus X Hereford reciprocal crosses) com­ pared with large frame cattle (Charolais, Gelbvieh, Main-Anjou and Brown Swiss X Angus or Hereford). They also observed greater activity of NADP-isocitrate dehydrogenase in subcutane­ ous adipose tissue of large frame cattle, but observed no difference in activities of acetyl-CoA carboxylase, glucose-6- phosphate dehydrogenase and 6-phosphogluconate dehydrogenase between the cattle of the two frame sizes. Thus, it is not clear whether fatty acid synthetic capacity or lipogenic enzyme 98 activities differ among different frame sizes of cattle.

B. The Citrate Cleavage Pathway and Fatty Acid Synthesis from Lactate

Glucose has been demonstrated to be a poor precursor of fatty acids in ruminant tissues (Kleiber et al., 1955; Ballard and Hanson, 1967a). Ballard and Hanson (1967a) showed that glucose was used at lower rates for fatty acid synthesis in liver and adipose tissues of mature cattle and sheep compared with rat liver and adipose tissue. They also observed lower activities of enzymes involved in the citrate cleavage path­ way in sheep and cow liver and adipose tissues compared to rat liver and adipose tissue. Hanson and Ballard (1968) ob­ served that rates of fatty acid synthesis from glucose were greater in liver of fetal cattle than in liver of mature cattle and that activities of enzymes of the citrate cleavage pathway were greater in fetal liver of cattle. The results of studies by Ballard and Hanson (1967a) suggested that the low rates of fatty acid synthesis from glucose in tissues of mature ruminants was because of reduced rates of carbon flow through the citrate cleavage pathway.

The hypothesis that reduced carbon flow through the citrate cleavage pathway explains poor rates of fatty acid synthesis from glucose in ruminant adipose tissues predicts that neither lactate nor pyruvate should serve as fatty acid precursors. 99

Whitehurst et al, (1978) , however, observed that rates of fatty acid synthesis from lactate and pyruvate were greater

than that from glucose in bovine adipose tissue and also ob­ served that rates of fatty acid synthesis from lactate were

similar to those from acetate in the presence of glucose.

Prior (1978) reported that lactate was used for carcass lipid

formation by sheep 40% to 70% as well as acetate depending upon

the dietary energy intake. Prior and Jacobson (1979b) have shown that both pyruvate and lactate are decarboxylated during

conversion to fatty acids by bovine adipose tissue. In the

present study, both lactate and acetate were used at similar rates for fatty acid synthesis in subcutaneous, intermuscular

and intramuscular adipose tissues of growing cattle, which

further confirms observations by Whitehurst et al. (1978). In

addition, acetate and lactate were used at similar rates for

fatty acid synthesis and at rates greater than that of glucose

for fatty acid synthesis in perirenal adipose tissue of sheep. The latter result confirms findings by Prior (1978) that lactate

can serve as a fatty acid precursor in sheep. These observa­

tions about lactate and pyruvate use as a fatty acid precursor

in ruminant adipose tissues contradict predictions based upon

explanations for poor glucose use as a fatty acid precursor and raise the question as to whether the citrate cleavage pathway

functions in ruminant adipose tissues. 100

To determine whether sufficient ATP-citrate lyase activity existed to support fatty acid synthesis from lactate, fatty acid synthetic rates from lactate and activities of ATP-citrate lyase were compared in growing cattle. In all adipose tissue depots studied, more than sufficient activity of ATP-citrate lyase was observed than was needed to support observed rates of fatty acid synthesis from lactate in adipose tissues of cattle.

This result suggested that lactate could be used for fatty acid synthesis via the citrate cleavage pathway and that some other enzymatic activity limited fatty acid synthesis from lactate in adipose tissue of cattle.

To determine which enzymatic reaction limits fatty acid synthesis from lactate, activities of acetyl-CoA carboxylase and NADPH-generating enzymes (glucose-6-phosphate dehydrogenase,

6-phosphogluconate dehydrogenase and NADP-isocitrate dehydro­ genase) were compared with rates of fatty acid synthesis from lactate in adipose tissues of growing cattle. In subcutaneous, intermuscular and intramuscular adipose tissues of cattle, considerably greater NADPH-generating potential was observed than needed to support fatty acid synthesis from lactate. In subcutaneous and intermuscular adipose tissues of growing cattle, mean rates of fatty acid synthesis and mean activities of acetyl-CoA carboxylase were similar. This result is con­ sistent with the proposed rate-limiting role of acetyl-CoA carboxylase in fatty acid synthesis (Lane et al., 1979), and 101 suggested that fatty acid synthesis from lactate is limited by activity of acetyl-CoA carboxylase in adipose tissues of cattle. To further confirm that the citrate cleavage pathway functions to support fatty acid synthesis from lactate, con­

version of propionate, glutamate and lactate were compared in subcutaneous bovine adipose tissue. Both glutamate and lac­ tate were used at similar rates for fatty acid synthesis and

at greater rates than for propionate. Glutamate labelled in the number 5 carbon atom can only be used for fatty acid synthesis via the citrate cleavage pathway (Leveille and Hanson, 1966a, b), and, thus, the use of glutamate for fatty acid syn­

thesis suggested that the citrate cleavage pathway can function

to support fatty acid synthesis from lactate in bovine adipose tissue.

Propionate also should be used for fatty acid synthesis

via the citrate cleavage pathway; however, propionate was not

utilized for fatty acid synthesis. At a concentration of

10 mM, propionate was oxidized to CO2 at approximately 50% the rate of oxidation of lactate or glutamate. This observa­

tion that propionate was oxidized to CO2 but not converted to

fatty acids agrees with observations by Hood et al. (1972). Because other results of the present studies also indicated

that the citrate cleavage pathway does function in bovine

adipose tissue, some other metabolic limitation must account 102

for the low rates of propionate conversion to fatty acids.

The limitation for propionate use as a fatty acid precursor

is unclear.

C. The Limitation for Glucose Use as a Fatty Acid Precursor

As previously discussed, the low rates of glucose con­

version to fatty acids had been explained on the basis of

reduced rates of acetyl-CoA formation via the citrate cleavage pathway. Results of studies previously discussed, however,

indicated that the citrate cleavage pathway does function in

bovine adipose tissue. Smith (1975) observed greater rates

of fatty acid synthesis from pyruvate than from glucose in

rabbit adipose tissue and suggested that low rates of glucose

conversion to fatty acid was because of low activity of

pyruvate kinase. Ballard and Oliver (1964) observed low activ­ ity of glucokinase in liver of cattle compared with activity

observed in rat liver. Raggi et al. (1963) also have observed

low glucokinase activity in adipose tissue of cattle compared

with activity in rat adipose tissue. Thus, it seemed possible

that either reduced phosphorylating ability or reduced flow

through the glycolytic pathway might account for the low rates

of glucose conversion to fatty acids in ruminant adipose tissues.

To determine possible limiting step in the catabolism of

glucose, glucose catabolism in bovine and ovine adipose tissues 103 was quantitated. Catabolism of glucose via the pentose phos­ phate pathway accounted for less than 10% of the total glucose catabolism in bovine and ovine adipose tissues. This result is lower than the values of approximately 20% estimated for rat adipose tissues (Katz et al., 1966; Landau and Katz, 1964).

In the present studies, glucose converted to CO2, fatty acids and glycerol accounted for less than 30% of the total glucose catabolized. In adipose tissue of ad libitum-fed rats, Katz et al. (1966) observed that CO^, fatty acids and glycerol accounted for between 71% and 86% of total glucose metabolized.

Yang and Baldwin (1973) observed that 50% of glucose catabo­ lized by isolated bovine adipocytes was accounted for by lac­ tate and pyruvate. Lactate and pyruvate formation from glucose were not quantitated in the present studies; however, it does appear that glucose metabolism is "altered" in ruminant adi­ pose tissues. The metabolic basis for this altered metabolism of glucose, however, is not apparent.

Rates of glucose uptake observed in bovine and ovine adi­ pose tissues were 25% to 35% of those observed in rat adipose tissues (Katz et al., 1966; Flatt and Ball, 1964). This result is consistent with the reported lower glucokinase activity in bovine adipose tissue (Raggi et al., 1963). In a recent report. Smith and Prior (1980) have suggested that glucose conversion to fatty acids might be limited by either hexokinase or pyruvate kinase. Because studies by Yang and Baldwin (1973) 104

indicated that substained catabolism of glucose to lactate and pyruvate occurs in isolated bovine adipocytes, it would appear that pyruvate kinase does function in ruminant adipose tissues. The low rates of glucose uptake observed in the present study and the reduced rates of glucose phosphoryla­ tion reported in studies of Raggi et al. (1963) and of Smith and Prior (1980) suggest that the low rates of glucose con­ version to fatty acids observed in ruminant adipose tissues is due either to reduced uptake or phosphorylation of glucose.

Additional work is needed to resolve whether ability to trans­ port glucose across the plasma membrane of fat cells or ability to phosphorylate glucose once inside the fat cell limits glu­ cose conversion to fatty acids in ruminant adipose tissues.

D. The Distribution of Adipocyte Sizes

Hirsch and Gallian (1968) introduced a method for counting and determining volumes of adipocytes that involved fixing adipocytes with osmium tetroxide and then counting and deter­ mining their volumes with an automatic particle counter. Sjostrom et al. (1971) presented evidence that the distribution of adipocyte sizes determined with an automatic particle coun­ ter was described by a normal distribution function. Some workers, however, have observed adipocyte size distributions that are skewed (Hood and Allen, 1975a; Hood and Thornton,

1979), and this observation suggested that adipocyte size 105 distributions might be described more adequately by some other distribution function.

In the present investigation, lognormal and normal models were compared for describing size distributions of bovine adipocytes. Both normal and lognormal models provided simi­ lar estimates of mean adipocyte size; the lognormal model, however, generally provided a better fit to particle counter data as evidenced by the lower values of (chi square) ob­ served. This result suggested that a lognormal distribution function more adequately describes adipocyte size distribution than does a normal distribution function. An advantage of using the lognormal distribution function was the ability to model distributions of adipocyte sizes with varying degrees of skewness. 106

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VIII. ACKNOWLEDGEMENTS

I would like to thank Dr. Donald C. Beitz for his guidance of my graduate studies. I also would like to thank him for his encouragement, patience and help with my research program. I would like to thank Dr. Paul N. Hinz, Dr. Richard

M. Robson, Dr. Carl L. Tipton and Dr. Jerry W. Young for serving on my committee. I also would like to thank Dr. David

G. Topel and Dr. David F. Cox for their help and suggestions. Special appreciation is expressed to Dr. Richard W. Willham for providing the experimental animals. Thanks also are expressed to Mr. Dennis Maxwell for caring for cattle while at the McNay Research Center. I also would like to thank

Mr. Randy Petersohn and Mr. Grant Gustafson for their help collecting samples at the Iowa State University Meats Labora­ tory.

I would especially like to thank Danilo Cianzio for his friendship, cooperation and assistance.

Special thanks also are extended to Ms. Diane Johnson for her help and cooperation with many parts of analytical work. I also would like to thank Mr. Paul Chace and Mr. Paul Smith for their technical assistance. In addition, I would like to thank the members of the Nutritional Physiology Group for taking time from their own projects to provide assistance when needed. Financial assistance from the Iowa Beef Industry 123

Council was greatly appreciated.

And finally, I would like to thank my parents, sister and brother for their support and encouragement. 124

IX. APPENDIX A

Analysis of variance for lactate and acetate conversion to fatty acids during growth of two biological types of cattle

Subcutaneous Adipose Tissue Mean Square Source DF Lactate Acetate Type 1 871344 268549 Age 3 97288413** 56263385 Type X Age 3 1242542 17940957 Error 24 16360671 24871014

Intermuscular Adipose Tissue Mean Square Source DF Lactate Acetate Type 1 139159 2773231 Age 3 16907018** 5853078 Type X Age 3 657181 3431281 Error 23 3371251 2736502

Intramuscular Adipose Tissue Mean Square Source DF Lactate Acetate Type 1 7118 13577 Age 3 4597511** 430159** Type X Age 3 15987 6500 Error 23 315405 66160

** P < ,01. 125

X. APPENDIX B

Analyses of variance for acetyl-CoA carboxylase and ATP- citrate lyase activity during growth of two biological types of cattle

Subcutaneous Adipose Tissue

Mean Square Source DF CBX^ CCE^ Type 1 187 208 Age 3 604 88784** Type X Age 3 8 24415 Error 24 328 17798

Intermuscular Adipose Tissue Mean Square Source DF CBX CCE Type 1 999 415 Age 3 1516 46440 Type X Age 3 1113 2211 Error 23 756 10733

Intramuscular Adipose Tissue Mean Square

Source DF CBX CCE Type 1 205 19007 Age 3 857 313976 Type X Age 3 1955 73498 Error 23 3611 67489

^Acetyl-CoA carboxylase.

^ATP-citrate lyase

**P < .01. 126

XI. APPENDIX C

Analyses of variance for glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-isocitrate dehydro­ genase activity during growth of two biological types of cattle

Subcutaneous Adipose Tissue Mean Square Source DF G6PDH 6PGDH ICDH^ Type 1 176696419 51773576 5360437 Age 3 1162650067** 142887641 309872438** Type X Age 3 145251709 33951667 24548505 Error 24 175655808 36152358 42991057

Intermuscular Adipose Tissue Mean Square Source DF G6PDH 6PGDH ICDH Type 1 99241946 3182251 1978869 Age 3 203781778 26173642 144967525** Type X Age 3 36694381 4872531 7761332 Error 23 54470225 6775118 25945591

Intramuscular Adipose Tissue Mean Square Source DF G6PDH 6PGDH ICDH Type 1 33270077 909829 1732000 Age 3 107310246 7298937 39031651** Type X Age 3 14135404 484859 19866603 Error 23 25755920 2347613 4760256

^Glucose-6-phosphate dehydrogenase. ^6-Phosphogluconate dehydrogenase.

"^NADP-isocitrate dehydrogenase. **P < .01. 127

XII. APPENDIX D

Simple correlation coefficients of lipogenic and enzymatic activities in subcutaneous adipose tissue

Item ACE^ LAC^ ACC° CCE^ G6P® PGD^ IDH^

ACE 1.00 LAC .37* 1.00 ACC .23 .46* 1.00 CEE .47* .09 .26 1.00 G6P .47* .25 .34 .76* 1.00 PGD .24 .19 .28 .66* .91* 1.00 IDH .53* .38* .28 .71* .85* .78* 1.00

^Acetate conversion to fatty acid. ^Lactate conversion to fatty acid.

^Acetyl-CoA carboxylase activity.

^ATP-citrate lyase activity.

®Glucose-6-phosphate dehydrogenase activity.

^6-Phosphogluconate dehydrogenase activity ^NADP-isocitrate dehydrogenase activity.

*Correlation coefficients significantly different from 0 (P < 0.05). 128

XIII. APPENDIX E

Simple correlation coefficients of lipogenic end enzymatic activities in intermuscular adipose tissue

Item ACE^ LAC^ ACC^ CCE^ G6P® PGD^ IDH^

ACE 1.00

LAC .36* 1.00 ACC .46* .10 1.00

CCE .08 .04 .10 1.00

G6P .30 .18 .27 .41* 1.00

PGD .24 .18 .30 .19 .76* 1.00

IDH .30 .41* .36* .15 .79* .63* 1.00

^Acetate conversion to fatty acid. ^Lactate conversion to fatty acid.

^Acetyl-CoA carboxylase activity.

^ATP-citrate lyase activity. ®Glucose-6-phosphate dehydrogenase activity.

^6-Phosphogluconate dehydrogenase activity.

^NADP-isocitrate dehydrogenase activity. *Correlation coefficients significantly different from 0 (P < 0.05). 129

XIV. APPENDIX F

Simple correlation coefficients of lipogenic and enzymatic activities in intramuscular adipose tissue

Item ACE^ LAC^ ACC^ CCE^ G6P® PGD^ IDH^

ACE 1.00

LAC .59* 1.00 ACC .27 -.07 1.00

CCE .39* .51* .24 1.00

G6P .64* .59* .34 .57* 1.00

PGD .41* .28 .24 .23 .50* 1.00

IDH .71* .77 .15 .56* .79* .44* 1.00

^Acetate conversion to fatty acid.

'^Lactate conversion to fatty acid.

^Acetyl-CoA carboxylase activity.

^ATP-citrate lyase activity. ®Glucose-6-phosphate dehydrogenase activity.

^6-Phosphogluconate dehydrogenase activity.

^NADP-isocitrate dehydrogenase activity.

*Correlation coefficients significantly different from 0 (P < 0.05). - ^ TT

MICROFILMED - 1981 O!

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