Efleds of Age, Pregnancy, and Lactation on Rat, Guinea Pig, and Cow Adipose Enzyme Activities and Cow Adipose Metabolism 1

R. L. BALDWIN, J. R. REICHU, S. LOUIS, N. E. SMITH 3, Y. T. YANG, and ELBA OSBORNE

Department of Animal Science, University of California, Davis 95616

Abstract

In the enzyme study, virgin, pregnant, lactating, and adult animals whose ages were comparable to lactating animals were compared. With all three species, young virgin adipose enzyme activities were higher than in older groups indicat- ing a prominent age effect. Comparison of adipose enzyme activities of adult and lactating groups indicated that activities of only a few enzymes were increased during lactation and some differences in response exist among species. In the general study of cow adipose metabolism through tissue slices, relative rates of glu- cose-l-, -2-, and -6-14C oxidation and in- corporation into glyceride glycerol were 1.0, .5, and .25 and .62, .92, and 1.0, indi- cating high hexose monophosphate shunt activity and high rates of glucose utiliza- tion for glyceride glycerol synthesis. Ratio of glucose-U-14C oxidation to use for lipogenesis was about 1.2, was increased by insulin in vitro, and was decreased in lactating as compared to nonlactating animals. Rates of glucose and acetate oxidation and use for lipogenesis were doubled approximately during lactation. Comparisons of rates of glucose oxidation with rates of fatty acid synthesis from acetate indicated that NADPH_~ yields from hexose-P oxidation via pentose cycle are not sufficient to support fatty acid synthesis. Hence, additional NADPHe generating reactions must occur. Rates of fatty acid esterification in adipose tissue slices exceeded rates of fatty acid synthe- sis by about four-fold presumably re- flecting extensive fatty acid reesterifica- tion.

Received for publication March 27, 1972. a Supported in part by USPHS grant AM07672. 2 Present address: Department of Animal Nutri-

tion, Hohertheim University, 7000 Stuttgart 70, Germany.

Present address: Department of Dairy Science, Comell University, Ithaca, New York 14850.

Introduction

Several years ago, Bartley et al. (6) report- ed a study of changes in specific activities of several enzymes associated with lipogenesis in mouse adipose, liver, and mammary tissues during pregnancy and lactation. In general, they found that specific activities of ghicose- 6-P dehydrogenase, malic enzyme, and citrate cleavage enzyme were higher in adipose, liver, and mammary tissues from lactating as com- pared to pregnant mice. This is consistent with the concept that liver and adipose tissue con- tribute to milk fat synthesis by converting ab- sorbed nutrients to fatty acids which are re- leased and eventually incorporated into milk triglycerides by the mammary gland (3). However, results of Bartley et al. (6) were not as simple as generalization implies. Tissue en- zymes were often lower in pregnant as com- pared to virgin animals. Increases during lac- tation did not always offset this depression so that activities of some enzymes were low- er, some similar, and some higher in virgin as compared to lactating mouse tissues. In addi- tion, activity of ghicose-6-P dehydrogenase in adipose tissue was lower in lactating than in virgin or pregnant mice. The complexity of these data wherein activities of some lipogenie enzymes increase and others decrease in ac- tivity with changes in physiological state pre- cludes generalization and dearly indicates that further studies of effects of pregnancy and lac- tation upon adipose lipogenesis are required.

Analyses of enzyme data in studies directed at different objectives but relevant to role of adipose tissue in lipid metabolism during lacta- tion also indicate that a complex series of en- zyme changes may occur in adipose tissue dur- ing lactation (1, 3, 4, 11, 15). Because of the above observations, the central role played by adipose tissue in lipid synthesis in ruminants (4), and indications that changes in adipose tissue metabolism in lactating cows may be partially responsible for low milk fat syndrome (9), studies reported herein were undertaken.

The first stage of our study was directed at extension of observations of Bartley et al. (6) to several additional species in hope that adi-

340

ADIPOSE METABOLISM 341

pose enzyme changes during lactation could be clarified. The second stage involved investi- gation of metabolism of cow adipose tissue slices in vitro as affected by pregnancy, lacta- tion, a milk-fat depressing diet, chronic insulin injection in rive, and insulin in vitro. Objec- tives of this stage were to characterize general metabolic patterns of cow adipose tissue, de- termine the relationship between adipose en- zyme patterns and metabolism in cow adipose tissue, and determine whether cow adipose metabolism is altered during lactation or by dietary and hormonal treatments.

Experimental Procedures Animals. Four groups each of rats, guinea

pigs, and cows were sampled in the interspe- cies study of effects of physiological state on adipose tissue enzymes. The virgin (90 days old), pregnant (100 to 110 days old, 15 days pregnant), lactating (120 to 130 days old, lac- tating 15 days), and adult virgin (130 to 140 days old) Sprague-Dawley rats from our col- ony were housed in plastic cages on wood shavings at 23 C. They were fed Purina Labora- tory Chow ad libitum and provided fresh water daily. In designing this study, we considered the possibility that comparisons of adipose en- zyme activities in animals differing in physi- ological states as widely as those we investi- gated would be complicated by differences in food intake and energy status. Resolution of the dilemma in selection of a suitable feeding regime and interpretation of data involved rec- ognition that differences in food intake and en- ergy status are components of the difference among several physiological states and should not be controlled.

From this arose the decision to feed ad libi- tum such that food intakes characteristic of each physiological state would be attained to interpret differences as being attributable to physiological state only and not attempt to speculate whether differences in enzymes were attributable to differences in food intake, ener- gy status, hormonal status, or any other com- ponents of difference between physiological status. Identification and evaluation of these factors were considered appropriate for future experiments but not the current, simple survey directed at determining whether differences exist.

Rats were killed by decapitation, and ex- cised abdominal adipose tissue was frozen im- mediately and stored for short periods in liq- uid nitrogen. Virgin (80 to 83 days old), preg- nant (130 to 140 days old, 50 to 55 days preg- nant), lactating (140 to 150 days old, lactating

3 days), and adult (135 to 140 days old) mixed colored guinea pigs from our colony were in the study. Guinea pigs were housed in wire cages with free access to water and Purina guinea pig chow and were fed lettuce once daily. Guinea pigs were killed by decapi- tation. Excised abdominal adipose tissue sam- ples were quick-frozen and stored in liquid ni- trogen until used.

Samples of cow abdominal adipose tissue were obtained at a local slaughter house. Rec- ords on about half the cows were not avail- able, and judgement was required in selecting animals for each group. Virgin heifers were in the range of 12 to 16 me old. Pregnant animal s were selected to be in the 7th to 8th me of their first pregnancy at about 2 yr of age. Lac- tating cows were in approximately mid-lacta- tion and 2 to 4 yr old. All cows were Holstein excepting one Guernsey and one Milking Shorthorn in the virgin and pregnant groups, respectively. Cow tissue samples were trans- ported to the laboratory on ice and stored fro- zen in liquid nitrogen until used.

Forty-eight cows were in the second stage of the study. Animals assigned to experimental groups provided for the following compari- sons: nonpregnant, nonlaetating Holsteins vs. nonpregnant, nonlactating Jerseys; nonpreg- nant, nonlaetating Jerseys vs. pregnant, non- lactating Jerseys; pregnant, nonlactating Jer- seys vs. lactating Jerseys vs. lactating Hol- steins; and lactating cows fed a normal herd ration of 405 concentrate, 605 alfalfa (G) vs. lactating cows fed the same diet but in- jected with 750 to 1,000 units of protamine zinc insulin daily for 4 wk (I) vs. lactating cows fed a 100% concentrate ration to reduce milk fat percentage (D).

Data regarding milk production and milk fat percentage in the latter three groups of lac- tating cows are in Table 1. The high concen- trate diet decreased milk production and milk fat percerLtage while other treatments had no significant effects. The insulin treatment group was included to test the hypothesis (1, 4, 9) that insulin might depress milk fat production. The result is negative and will be discussed in detail elsewhere (17, 18).

Because no breed differences were observed, no distinctions as to breed were made in final analysis of data. To simplify presentation of data in cases where data on dry, nonpregnant and dry, pregnant cows did not differ, data were combined to form a group referred to as nonlactating. Also, when no differences due to treatment in lactating cows were observed,

JOURNAL OF DAIRY SCIENCE VOL. 56, NO, 3

342

TABLE 1. Milk production data)

BALDWIN ET AL

Treatment Milk production Milk fat group Period 1 Period 2 Period 1 Period 9.

(kg/day) (~) I (insulin) 17.3 +-- 2.1 17.2 +-- 2.3 4.5 ± .43 4.7 ± .41 C (normal) 13.5 - 1.6 ll.O ± 1.8 5.0 ± .42 5.~ ± .59` D (all conc.) 16.7 --- 3.2 11.4 _ 2.5 4.6 ± .46 2.8 _ .25

Treatment groups as described in text and footnote of Table 3. Period 1 data are for a 1 wk preliminary period before initiation of experiment when all cows were fed the control ration. Period 9, data are from the last wk of the 4 wk experimental period. Thus, milk production values are average daily production for 1 wk _+ SEM; Milk fat percentages were determined (Babcock) in 1 wk composite milk samples for each cow and are expressed as mean fat percentage ± SEM.

data were combined to form a group referred to as lactating cows.

Experimental (animal) variance is high in tissue studies with cows, and stated combina- tions of data did, in several cases, aid in dem- onstrating statistical significance.

Enzymatic measurements. Assays of the en- zymes were in mitochondria free supernatants (20,000 X g, 20 min) with methods and pre- cautions detailed in several previous publica- tions (1, 2, 11, 12).

Tissue slice studies. Basic methods in tissue slice experiments were essentially as described by Bartley et al. (5). Tissue slices were pre- pared from samples of adipose tissue removed from the body wall of the internal thoracic area and forward of the rib cages of experi- mental cows within 30 to 45 rain of slaughter with a Stadie-Riggs hand microtome. For com- parison of activities of adipose tissue samples from different sites (Table 7), pefirenal and subcutaneous samples were collected from dry, nonpregnant cows. The subcutaneous site sam- pled was the fat pad above the rear udder at- tachment. Samples were held at 37 C between collection and slicing to prevent cell damage due to hardening of fat. One hundred milli- grams of tissue slices were incubated for 2 hr in a Dubnoff metabolic shaker at 37 C under a 95% oxygen, 5% carbon dioxide gas mixture in 25-ml siliconized Erlenmeyer flasks contain- ing 2 ml of incubation media previously equili- brated with the CO2:O z gas mixture. Incuba- tion flasks were stoppered with self-sealing rubber serum caps with plastic center wells at- tached. Incubations were stopped by injection of .5 ml of 1 N H2SO4 into the incubation me- dium. Hydroxy-hyamine (.25 ml) was added to the center well by injection with needle and syringe, and flasks were incubated with shak- ing for an additional 30 to 45 rain to allow trapping of CO2. The plastic center wells were then transferred to scintillation counting vials JOURNAL OF DAIRY SCIENC~ VOL. 56, ~N~O, 3

containing counting fluid for counting in a liq- uid scintillation counter.

Following collection of COz, total cell lipids were extracted by method of Dole (10). A portion of total lipid extract was air-dried and counted in 15 ml of scintillation counting fluid. The remainder was saponified with an excess of ethanol-KOH for 2 Itr at 70 C; the solution was then acidified with HC1 and fatty acids were extracted with hexane. This extract was air-dried mad radioactivity determined. Sub- traction of counts in fatty acids from counts in total lipid extract fielded an estimate of counts in glyceride glycerol (10). All incubation treatments were in triplicate.

The standard incubation medium was Krebs-Ringer bicarbonate buffer with half the recommended calcium concentration and 4.0% bovine serum albumin (fraction V), 30 rag / 100 ml of casein hydrolysate, and 1.0 mg/100 ml each of tryptophane and methionine added. Glucose, acetate, and albumin-palmitate were added to incubation medium at concentrations of 2.7, 2.5, and .53/anoles/ml. Insulin concen- tration, when added, was .5 milliunits/ml of incubation media. Radioactive tracers were added at concentrations between .1 and .3 /~curies/ml of incubation media. Glucose, ace- tate, albumin-palmitate, and amino acids were added to incubation media so comparisons be- tween cow treatment groups could be based upon results with tissue slices incubated with all major substrates normally present in vivo. Also, all substrates present were at concentra- tions comparable with those in blood and ex- tracellular fluids.

During the course of the study, possible ef- fects of deleting one or all unlabeled substrates from the incubation medium were investi- gated. The only significant effect was an in- crease in acetate oxidation when palmitate was deleted (Table 4). Metabolic interactions be-

ADIPOSE METABOLISM 343

TABLE 2. Effect of physiological state upon activities ol selected enzymes in rat, guinea pig, and cow adipose tissues.

Species and enzymes Physiological state ~

Adult Virgin Pregnant Lactating

Rat Body weight (g) 271 ± 2.6 226 - 7.2 Extractable protein (mg/g) 8.3 ± 1.2 8.3 ± 1.1 Glucose-6-P dehydrogenase .15"± .01 .38~___ .09. 6-P-Glueonate dehydrogenase .28 b± .02 .54 ~ + .01 Fructose-I, 6-dip aldolase .15b__- .01 .43~--- .03 a-Glycerol-P dehydrogenase

(NAD) 1.1 __- .12 2.5 ± .11 Isoeitrate dehydrogenase

(NADP) .48 b___ .03 .78 a ± .03 Malate dehydrogenase

(Cytoplasmic) 14.9, b ± .8 28.6 ~ ± .8 Malie enzyme (NADP) . l l b ± .O0 .34"± .04

Guinea pig Body weight (g) 759 ___41 685 231 Extractable protein (mg/g ) 5.7 ¢ ± .4 6.0 ¢ ± .3 Glueose-6-P dehydrogenase .20 ¢__- .02 .48 ~ - .03 6-P-Gluc(mate dehydrogenase .07 ~± .01 .09 ~_ .02 Fructose-l, 6-dip aldolase 1.2"E± .2 1.9 ~ ± .2 a-Glyeerol-P dehych-ogenase

(NAD) 4.2 ~ ± .6 6.(P ± .8 Isocitrate dehydrogenase

(NADP) .07~± .01 1.5 '~ --- .3 Malate dehydrogenase

(cytoplasmic) 38 ~ __- 2.3 140 ~ ± 6.8 Malic enzyme (NADP) .14b± .Ol .33"± .03

Cow Extractable protein (rag/g) 5.5 b ± .25 9.8 ~ ± .45 Glucose-6-P dehydrogenase .09b± .01 .22~± .02 6-P-Gluconate dehydrogenase .08 b± .01 .18" ± .02 Fructose-I, 6-diP aldolase .29~± .02 .53"± .03 a-Glyeerol-P dehydrogenase

(NAD) .28b± .01 .63~± .06 Isoeitrate dehydrogenase

(NADP) .84b± .03 1.98~± .10 Malate dehydrogenase

(cytoplasmic) 6.5 ~ ± .41 7.1 ~ ± .43 Malie enzyme ~NADP) .18~± .02 .14"--- .01

282 _____ 9.5 340 ± 14.1 7.1 ± 1.1 10!0 ± 1.7

.27~E± .06 .28b± .02

.32 b ± .05 .42b± .04

.12 ~ ± .00 .53"± .02

1.7 ± .08 2.3 ± .12

.81 ~ ± .06 .77"2 .04

33.2 a ± .8 35.0 ~ ± 1.9 .28"b± .08 .15~± .03

1,008 ± 66 16.9 ~ ± 2.2

.19.d ± .01

.01~ b ± .02

.70 ~ ± .08

717 260 26.4" ± 2.6

.28b± .01

.08 "± .01 1.6 ~ ± .10

2.4 ~ ± .3 1.3 ~ ± .1

.07 e 4- .01 .4 b _4- .1

33 ° ± 1.9 68 b ± 2.7 .16 b ----- .03 .16b± .01

5.3 b ___ .22 5.6 ~ ± .49 .05 c ± .00 .12b± .02 .07 b ± .0'1 .07 b± .01 .28 ° ± .02 .22~----- .02

.23 b ± .02 .28b± .0'3

.98 b _ .05 1.02b± .08

4.60 ~ ± .36 5.8 ~ - .44 .06 b ± .00 .05 b_ .01

x Abdominal adipose tissue enzyme activities expressed as/anoles converted per min per gram tis- sue under standard assay conditions.

a,b.c,a Different superscripts on horizontal indicate statistical differences (multiple range test; P < .05).

tween insulin, acetate, and glucose in young ruminan t adipose samples repor ted by Bartos and Skarda (7) were inves t iga ted in another exper iment and will be repor ted separate ly (17) .

Statistical analyses. T h e S tuden t -Newman- Keules mul t ip le r ange test was used to evalu- a te t r ea tment differences (16) .

Results and Discussion

Results of s tudying effects of physiological s tate on abdomina l adipose enzyme activit ies

in rats, guinea pigs, and cows are in Tab le 2. Ext rac tab le prote in was not affected apprecia- b ly by physiological state in the rat. Activi t ies of glucose-6-P dehydrogenase and 6-P-glu- cona te dehydrogenase were sl ightly depressed in p regnan t and lac ta t ing animals and clearly lower in adul t as compared to vi rgin animals. Aldolase and, possibly, a-glycerol-P dehydro- genase were depressed dur ing p regnancy and in the adul t group. Isoci t rate dehydrogenase and mala te dehydrogenase were lower in the adul t than in o ther groups. Malie enzyme was

JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 3

3 4 4 B A L D W I N E T AL

depressed in both lactating and adult rats. Enzyme activities tend to decrease with age

such that adult values are consistently lower. This trend apparently is partially, but not to- tally, retarded by pregnancy and lactation. These data raise a point regarding selection of c~ntrol animals for evaluating effects of lacta- tion on adipose enzymes. Concluding that adi- pose NADP-linked dehydrogenase activities are depressed in lactafiag animals would be reached if virgin and pregnant animals were used for comparison while the contrary conclu- sion is reached that activities of these enzymes in lactating rats are unaffected or increased ff adult rats similar in age to lactating rats are used as controls (Table 2). Care must be taken to assure that age effects are not con- fused with physiological changes associated with lactation.

Large differences in extractable protein were observed in the study of guinea pig adipose en- zyme activities (Table 2). This produces a dilemma in interpretation because it questions the validity of expressing enzyme data per gram tissue, because differences in extractable :~ protein could be interpreted as reflecting dif- ferences in amounts of fat per cell. Expression of enzyme activities per milligram protein, however, does not resolve the problem since o~ much extractable protein of adipose tissue is of extracellular origin. An alternate method of "~ evaluating or expressing enzyme data is on a relative basis, i.e. to select a reference enzyme such as malate dehydrogenase or aldolase whose activity is not usually affected by changes in physiological or hormonal states or diet and express other enzyme activities rela- .~ tive to its activity. In this case, it seems ap- propriate to bear in mind all three alternative = o means of expression in arriving at interpreta- o tions.

Glucose-6-P dehydrogenase, 6-phosphoglu- .~ conate dehydrogenase, and malic enzyme ac- = tivities per gram of tissue apparently were de- pressed during pregnancy and lactation rela- .~ t i re to virgin controls as observed with the rat. "~ This is emphasized if activities are expressed per milligram protein and effectively elimi- .~ nated if activities are expressed relative to al- dolase or malate dehydrogenase. As with rats, apparent effects of pregnancy and lactation "6 upon adipose enzymes associated with lipo- *5 genesis are most likely attributable to age rath- u~ er than pregnancy or lactation since enzyme ua activities in the adult group are similar to those 05

tfl in lactating group. These differences are not likely attributable to differences in food intake JOURNAL OF DAIRY SCIENCE VOL. 56, No. 3

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ADIPOSE METABOLISM 345

TABLE 4. Pattern of glucose oxidation, in vitro responses to insulin, and effect of palmitate on acetate oxidation?

N o n - Substrate Lactating lactating

Glucese-2-/-1-~'C ( + I ) .51-+.03 .50-+.02 Glucose-6-/-1-"C ( + I ) .26_ + .03 .23-+ .02 Glucose-l-l*C (+U- I ) 1.24-+.08 1.43-+.06 Glucose-2-~C ( + I / - I ) 1.14-+ .05 . . . Glueose-6-1*C ( + I / - I ) 1.32-+ .09 . . . Acetate-l-a'C (+p / -p ) .574-.07 . . .

Based on same data in Table 3. Analysis of pattern of glucose utilization was accomplished by calculating ratios of glueose~2-14C and glucese-6- a'C oxidation to glucese-l-~4C oxidation for indi- viduals and calculating standard errors of the mean ratio and testing vs. 1.0; all ratios were different from 1.0 (P<.001). Effect of insulin was evaluated by taking ratios of incubation with and without insulin and calctdating standard error of the mean ratios and testing vs. 1.0; all insulin responses were significant ( P<.01 ). Effect of deleting palmi- tate from incubation medium was evaluated by calculating ratios of acetate oxidatiort in presence and absence of palmitate, calculating the standard error of the mean ratio, and tesOng vs. 1.0. The effect was significant (P<.001).

or energy status, since the pattern of change is not consistent with changes in these two characteristics. Adult and lactating groups like- ly represent the two extremes of energy excess and energy deficit, yet have the most similar enzyme patterns. This was true for all three species. According to all criteria of expression and interpretation, a-glycerol-P dehydrogenase activity was depressed during lactation in guinea pigs. No such tendency was evident in rat adipose indicating a possible difference be- tween species.

Influence of age upon interpretation of en- zyme changes during pregnancy and lactation is clear in cow data (Table 2). Adipose en-

zyme activities in virgin animals of breeding age were almost uniformly higher than those during pregnancy and lactation and in the adult cow group. Malic enzyme activity often used as an indicator of lipogenic activity in nonmminant species (13) but not in rumi- nants (4, 8) was lower in adult nonpregnant, nonlactating cows. Data in subsequent tables (3 and 5) clearly indicates that adipose lipo- genesis increased during lactation in cows in the second stage of our investigation. Despite danger inherent in comparing enzyme data ob- tained with abdominal adipose samples from one group of cows with tissue slice data on lipogenesis obtained with internal thoracic adi- pose samples from a second group of cows, the possibility is implied by such a comparison (Table 1 vs. Tables 3 and 5) that increases in adipose lipogenesis during lactation were not reflected generally by increases in activities of enzymes studied (Table 1).

Previous studies (1, 11, 15) of enzymes in cows have consistently indicated that adipose enzyme activities are not elevated and may be depressed during lactation as in this study. En- zyme data on nonlactating cows in the second stage of this study and for which lipogenesis data are reported were low and erratic due to fatness of the cows. This excessive fatness is indicated by the low extractable protein values reported for several cows in Table 7. Because of this, enzyme data are not reported even though apparently consistent with speculation that increased adipose lipogenesis during lac- tation might not be attributable to general in- crease in lipogenic enzyme activities.

Results of this preliminary or survey study of adipose enzyme activities in several species and physiological states were presented be- cause they were considered confusing when collected and attempts were made to interpret data in terms of effects of physiological state upon adipose lipogenesis; because data led us

TABLE 5. Adipose Iipogenesis from glucose and acetate in nonlactating and lactating dairy cows?

Nonlaetating Lactating Substrate -- I + I -- I + I

Glueose-l-l*C 18.8b± 3.6 18.8 b - 3.1 33.4"----- 4.4 33.2"----- 3:0 Glucose-2-14C 21.T°-+ 9..2 22.2 b - 1.8 74.2"---11.8 73.8a--. 11.0 Glucose-6-'4C 21.0b----- 3.0 32.3 b - 8.7 60.6"-- + 9.7 83.3"-+ 17.5 Glueose-U-'~C 89.3b-+ 9.6 118.6b-+22.1 289 a -+41.7 346" ----- 44.5 Acctate-l-~'C 193 ~ -+33.0 '244 b -+42.2 419" -+67.1 565 a -+101.1

1 Values expressed in m#gatoms carbon from labeled position of substrate converted to product per 9. hr/100 nag tissuo slfces _ SEM. N for noulactating animals was 11 and for lactating animals was 21.

.,b Horizontal values with different superscripts are different (P<.05). JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 3

3 4 6 BALDWIN ET AL

to more intensive studies, reported subsequent- ly (18); because of several data indicate spe- cies differences in patterns of enzyme re- sponses to ehanges in physiological state; be- cause data illustrate a number of problems at- tendant with interpretation of enzyme data; and, most importantly, because data indicate necessity of using adult animals of comparable age in studies evaluating effects of lactation on metabolic parameters in extra-mammary tis- s u e s .

Effects of lactation and insulin in vitro on rates and patterns of substrate oxidations in cow adipose tissue slices are in Tables 3 and 4. Rates of oxidation of glucose-l, -2-, -6-, and -U-~C, acetate-l-~4C but not palmitate-1- x4C were lower in nonlactating than lactating cows. Rates of glucose -1- and -2-14C oxida- tion were not affected by lactating cow treat- ment. Glucose-6-14C oxidation appeared to be higher in cows fed high concentrate diet. Ace- tate oxidation was increased by insulin treat- ment in vivo. Data on glucose-U-a~C oxidation for lactating cows had a high variance, did not appear to indicate treatment effects, and, therefore, were pooled for analysis. Relative rates of glucose-l-, -2-, and -6-~4C oxidation were 1.0, .5, and .25 in cow adipose slices and this pattern was not affected by lactation (Ta- ble 4). These ratios indicating relative rates of oxidation of different glucose carbons sug- gest the major route of glucose oxidation in cow adipose tissue is the hexose monophos- phate pathway and considerable recycling via this pathway occurs. Ratios also reflect con- siderable diversion of glucose carbon to glyc- eride glycerol synthesis as indicated by data in Table 5. Bartos and Skarda (7) reported ratios for glucose-6-~4C/glucose-l-~4C oxida- tion of from .36 to .47 for omental adipose samples from 3 to 4 mo old goats. These val- ues are significantly higher than the ratio of .25 in this study. The difference may be due to species or to differences in the ages of ani- mals in the two studies. Responses in glucose oxidation to insulin added in vitro were all sig- nificant (P<.01) and ranged between 15 and 435 (Table 4). These magnitudes of response to insulin are consistent with previous reports of insulin actions upon glucose oxidation when acetate is included in incubation medium (7). Addition of palmitate to incubation media re- duced acetate oxidation 405 (Table 4), an effect significant both in presence and absence of glucose in the incubation media.

Rates of utilization of glucose and acetate for lipogenesis in lactating and noulactating cows are summarized in Table 5. More than JOURNAL OF DAIRY SCIENCE VOL. 26, NO. 3

95~; of glucose carbon incorporated into tis- sue lipids during incubation was recovered in the gtyceride glycerol fraction. Almost 1007o of acetate carbon incorporated into lipids was recovered in fatty acids. Hence, glucose in- corporation into lipids is an index of fatty acid esterification while acetate incorporation is an index of fatty acid synthesis. The data in Table 5 clearly indicate that triglyeeride synthesis in adipose tissue is increased two fold or more during lactation and fatty acid synthesis is in- creased similarly. Although there was a tend- ency for insulin in vitro to increase glucose-6- and -U-14C and acetate -1-1aC incorporations into lipids, trends were not statistically signifi- cant. Further analyses of data in Table 5 re- vealed a nonsignificant trend for insulin in vitro to decrease relative contributions of glu- cose-l- and -2-1~4C incorporation into lipid while glucose-6-14C incorporation increased. Also, lactation tended to decrease relative con- tribution of glucose-l-14C to glyceride glyc- erol, to increase relative contribution of glu- cose-2-1~C, and not to affect relative contribu- tion of glucose-6-1aC. These nonsignificant trends were in agreement with the slight changes in patterns of glucose oxidation. Mean incorporations of glucose-l-, -2-, and -6-1~C into glyeeride glycerol were .15, .22, and .24 of glucose-U-~4C incorporation and in sum ac- count for ~ of glyceride glycerol synthesis from ghicose-U-x4C, a seemingly high value. These values reflect well, however, the high usage of glucose for triglyceride glycerol syn- thesis.

The restatement and analysis of data from Tables 3 and 5 in Table 6 was constructed to provide ready evaluation of patterns of metab- olite utilization for oxidation and lipogenesis in cow adipose tissue; comparison of maximum rates of NADPHz formation and utilization; comparison of rates of fatty acid synthesis and esterifieation; and evaluation of possible con- tributions of glucose, acetate, and palmitate to adipose energy expenditures in vitro. In sev- eral of data transformations in Table 6, as- sumptions are critical and these will be stated for each case.

Data in columns labeled oxidation (OX) and lipogenesis (LIP) were directly from Tables 3 and 5. Ratios of oxidation to lipogenesis in the next column (OX/LIP) indieate that ap- proximately 2.2, .78, and .25 moles of glucose carbons 1, 2, and 6 are oxidized for each mole incorporated into glyceride glycerol. Overall OX/LIP ratio for glucose was about 1.2. This representation dearly indicates the prominence of hexose monophosphate pathway and of

TABLE 8. Analysis of data from

In

ADIPOSE METABOLISM 347

Tables 3 and 5 in patterns of nutrient utilization in adipose tissue.

Use for: Max. Fatty acid vitro NADPI-L esterified

Animal treat- yield or or synthe- Substrate status ment OX ~ LIP ~ OX/LIP use ~ sized a

(re#moles/2 hr) Glucose-l-~'C NL + I 46 -+ 9 18.8- + 3.1 2.4 +92 18.8

L + I 67 -+ 9 33 -+ 3 2.0 +134 33 Glucese-2-~'C NL + I 24 _+ 6 22 _+ 2 1.1 +48 22

L + I 33 -+ 5 74 -+ 11 .45 +66 74 Glueose-6-'4C NL + I 8.8_ + 2 32 -+ 7 .28 +17.8 32

L + I 18 -+ 2 83 -+ 18 .22 +36 83 Glucose-U-"C NL + I 158 _+23 119 _+ 22 1.31 +312 119

L + I 419 _+76 346 _+ 44 1.21 +838 346 NL --I 104 _+30 89 _+ 20 1.17 +208 89 L --I 297 -+44 289 _+ 42 1.02 +595 290

Acetate-l-a'C NL + I 35 _+ 5 244 _+ 42 .15 --427 30 L + I 109 _+17 565 -+101 .19 --1,072 77 NL --I 37 _+ 5 19'3 _+ 33 .18 --337 24 L --I 93 _+19 419 -+ 67 .22 --734 52

Palrnitate-l-:'C NL qrI 1.6-+ .6 . . . . . . . . . . . . L + I 1.7-+ .2 . . . . . . . . . . . .

a OX and LIP indicate m~gatoms labeled carbon incorporated into CO2 and fatty acids, re- spectively, by 100 mg tissue slice in 2 hr.

NADPH~ yield calculated for glucose-~'C on basis of two NADPH~ formed per glucose carbon con- verted to x~CO~. NADPH~ use calculated for acetate based upon 14 NADPH~ per eight aeetate-l-~'C in- corporated into fatty acids.

3 One fatty acid esterifiecl per glucose carbon incorporated into glyceride glycerol and one fatty acid esterified per eight aeetate-a*G incorporated into triglyceride fatty acids.

triose-P use for glyceride glycerol synthesis. There appears a consistent (all carbons) tend- ency for lactation to decrease the OX/LIP ra- tio, i.e. to increase the relative rate of glucose use for lipogenesis as compared to oxidation. Acetate oxidation appears to be about 20% of acetate use for lipogenesis and to increase slightly relative to lipogenesis during lactation. Insulin in vitro tends to decrease OX/LIP for glucose and increase OX/LIP for acetate.

Calculations leading to estimates of maxi- mum NADPH2 yield and use involved several assumptions. In glucose oxidation to provide NADPH2, oxidation of one glucose carbon to a4CO 2 was assumed to yield two NADPH2 in- dependent of position of the carbon in glucose. This assumption is most likely invalid but yields a maximum estimate, In acetate use for fatty acid synthesis, it was assumed that 14 NADPH2 were required for incorporation of 8 acetate (acetate-l-14C) units. In nonlactat- ing cows, oxidations of glucose carbons -1-, -2-, and -6- provided 30, 16, and 6~ of total NADPH2 that could be formed from glucose. Comparison of values for maximum NADPH2 formation from glucose (-U-14C) oxidation with estimates of the amount of NADPH2 re- quired for fatty acid synthesis from acetate in-

dicates that, under no condition, could glucose oxidation satisfy the NADPH2 requirement for fatty acid synthesis. For example, in adipose tissue from nonlactating animals incubated with insulin, a maximum of 312 m/~moles of NADPH2 could be formed from glucose and 427 mbanoles NADPH 2 are required for fatty acid synthesis from acetate. These computa- tions indicate that 23~; or more of NADPH2 required for fatty acid synthesis in cow adipose tissue must be derived from oxidative reactions not involving glucose. This observation is con- sistent with the suggestion of Bauman et al. (8) for cow mammary tissue and Yang and Baldwin (17) for cow adipocytes that oxida- tion of isoeitrate by cytoplasmie NADP linked isoeitrate dehydrogenase is a significant source of NADPI-Iz required in a-aminant tissues for fatty acid synthesis.

Assumptions in calculating glyceride glyc- erol formation from glucose and amount of glucose required for esterifieation of newly Synthesized fatty acids (Table 6) were: in- corporation of I m/~gatom of glucose-U-~4G carbon into glyceride glycerol reflected esteri- fieation of I m/xmole of fatty acid and incor- poration of 8 mt~gatoms of acetate-l-~aC into fatty acids reflects synthesis of 1 mtmaole of

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348

TAULE 7. Comparison of adipose sites.

BALDWIN ET AL

Adipose site ~ P T S

Glucose oxidation "~ 1.0 b ± .0 Acetate oxidation 2 1.0 u ± .0 Palmitate oxidation ~" 1.0 b ± .0 Lipogenesis from acetate "~ 1.0 b __+ .0 Malate dehydrogenase ~ 2.5 ~ ± .3 Isocitrate dehydrogenase ~ .40 b ± .14 Glucose-6-P dehydrogenase plus 6-P gluconate dehydrogenase 2'~ Extractable protein (rag/g)

4.1 ~ -- 1.3 6.2 ~ --+ 1.9 2.3 ~ ± .5 2.6 ~ ± .6 1.2 ~b ± .1 1.38 ± .3 2.9 --- .6 2.9 ± .4 2.5 ~ _--. .5 3.9 ~ ± .4

.83 b __. .25 2.24" ± .96

. 0 6 b ----_ .02 .04 b --- .02 . 2 9 a ± .10

.92 ± .30 2.1 ± .55 4.9 ----- 2.0

Samples were perirenal adipose, (P), adipose adhering to the body wall in internal thoracic area (T), and subcutaneous adipose (S) removed from fat pad above rear of udder.

Values expressed on relative basis with activity of perirenal adipose set to 1.0. Enzyme activities expressed as gmoles product form per min per gram tissue.

~,b Horizontal values with different superscripts are different (P < .05).

fat ty acid requiring esterification. On this basis, (Table 6) four to six times more glucose carbon is incorporated into glyceride glycerol than is required to esterffy newly synthesized fatty acids. This excessive incorporation is pre- sumed to reflect reesterification of fatty acids released in tissue due to lipase action and will be discussed in greater detail in a subsequent publication (18).

During the course of the cow study, com- parison was made of activities of perirenal in- ternal thoracic and subcutaneous adipose sam- ples from dry, nonpregnant cows. l~esults are summarized in Table 7. From the comparison, perirenal adipose was least active in oxidizing glucose, acetate, and palmitate and in syn- thesizing fatty acids from acetate; subcutane- ous fat was generally most active; and fat from the internal thoracic area was intermediate in activity (Table 7) . The greatest differences b y site were in rates of glucose oxidation which were six times higher in subcutaneous than perirenal fat. Malate dehydrogenase activity was similar in adipose samples from all three sites, while activities of isocitrate dehydroge- nase (NADP) and hexose phosphate dehydro- genases were five times higher in subcutane- ous than perirenal adipose samples. There was no parallel between enzyme activities and rates of substrate oxidation or lipogenesis. In in- tenaal thoracic compared to perirenal adipose, enzyme activities were similar while rates of glucose and acetate oxidation were 4.1 and 2.3 times greater, and lipogenesis 2.9 times greater in internal thoracic samples.

References

(1) Baldwin, R. L., H. H. Lin, W. Cheng, R. Cabrera, and M. Ronning. 1969. Enzyme and metabolite levels in mammary and

JOURNAL OF DAIRY SCIENCE VOL. 5 6 , No. 3

abdominal adipose tissue of lactating dairy cows. J. Dairy Sei., 52:183.

(2) Baldwin, R. L., and L. P. Milligan. 1966. Enzymatie changes associated with the in- itiatien and maintenance of lactation in the rat. J. Biol. Chem., 241:2058.

(3) Baldwin, R. L., and N. E. Smith. 1970. In- termediary aspects and tissue interactions 'of ruminant fat metabolism. ]. Dairy Sei., 54:583.

(4) Ballard, F. J., R. W. Hanson, and D. S. Krenfeld. 1969. Gluconeogenesis and lipo- genesis in tissue from nmfinant and non- ruminant animals. Fed. Proe., 9_8:218.

(5) Bartley, ]. C., S. Abraham, and I. L. Chai- koff. 1966. Biosynthesis of lactose by mam- mary gland slices from the lactating rat. J. Biol. Chem., 241:1132.

(6) Bartley, J. C., S. Abraham, and I. L. Chai- koff. 1966. Activity patterns of several en- zymes of liver, adipose tissue, and mam- mary gland o~ virgin, pregnant and lactat- ing mice. Proc. Soc. Exp. Biol. Med., 123: 670.

(7) Bartos, S., and J. Skarda. 1970. The effect o f insulin and acetate on the metabolism of glucose-U-14C in the adipose tissue of the goat. Physiologa Bohemoslovaea, 19:139.

(8) Bamnan, B. E., R. E. Brown, and C. L. Davis. 1970. Pathway of fatty acid synthe- sis and reducing equivalent generation in mammary gland of rat, sow and cow. Arch. Bioehem. Biophys., 140:237.

(9) Davis, C. L., and R. E. Brown. 1970. Low- fat milk syndrome. Page 545 in Physiology of digestion and metabolism in the rumi- nant, A. T. Phillipson, ed. Oriel Press Ltd., New Castle upon Tyne, England.

(10) Dole, V. P. 1956. A relation between non- esterified fatty acid in plasma and the me- tabolism of glucose. ]. Clin. Invest., 35: 150.

ADIPOSE, METABOLISM 3 4 9

(11) Howarth, R. E., R. L. Baldwin, and M. Ron- ning. 1968. Enzyme activities in liver, mus- cle, and adipose tissue of calves and steers. ~. Dairy Sci., 51:1270.

(12) Korsrud, G. O., and R. L. Baldwin. 1972, Ef- fects of adrenalectomy, adrenaleetomy-ovari- ectomy and eortisol and estrogen therapies upon enzyme activities in lactating rat mammary glands. Canadian J. Biochem., 50:366.

(13) Leveille, G. A. 1970. Adipose tissue me- tabolism: influence of periodicity of eating and diet composition. Fed. Proe., 29:1294.

(14) O'Hea, E. K., and G. A. Leveille. 1969. Significance of adipose tissue and liver as sites of fatty acid synthesis in the pig and

the efficiency of utilization of various sub- strates for lipogenesis. J. Nutr., 99:338.

(15) Shirley, J. E., R. S. Emery, E. M. Convey, and W. D. Oxender. 1971. Enzymatic changes in bovine adipose and mammary tissue with initiation of lactation. J. Dairy Sci., 54:780. (Abstr.)

(16) Sokal, R. R., and F. J. Rohlf. 1969. Pages 239 and 687 in Biometry. N. H. Freeman and Co., San Francisco.

(17) Yang, Y. T., and R. L. Baldwin. 1973. Preparation and metabolism of isolated ~lls from bovine adipose tissue. J. Dairy Sci., 56:350.

(18) Yang, Y. T., and R. L. Baldwin. 1973. Lipolysis in isolated cow adipose ceils. J. Dairy Sci., 56:366.

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