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Enzyme and Metabolite Levels in Mammary and Abdominal Adipose Tissue of Lactating Dairy Cows1, 2
Enzyme and Metabolite Levels in Mammary and Abdominal Adipose Tissue of Lactating Dairy Cows R. L. BALDWIN, H. J. LIN, W. CHENG, R. CABRERA, and hi. RONNING
Department of Animal Science, University of California, Davis Abstract
production in the rumen (15, 18, 19), of changes in the patterns of f a t t y acids in milk f a t (14, 19), of alterations in blood metabolites (17), and of indications of metabolic adaptations in adipose and mammary tissue (13, 19). Our study was undertaken to confirm a previous observation that marked changes in the levels of several enzymes occur in adipose tissues of cows fed high concentrate diets (13, 14) and to attempt to ascertain the metabolic significance of these changes.
Compared to hay, when concentrates were fed three- to fourfold, increases occurred in the activities of enzymes associated with f a t t y acid synthesis and esterification in adipose tissue, whereas mammary enzyme levels were relatively unaffected. The proportion of stearie acid in milk fat, presumably arising from adipose tissue, was significantly depressed in the high response group (severe milk fat depression) but not in the low response group (moderate milk f a t depression). Differences in dietary effects upon mammary intermediary metabolite levels between the high and low response groups suggested a relatively lower availability of energy to mammary glands of cows in the high response group when fed concentrate. A number of changes in adipose tissue metabolite levels were noted in concentratefed animals, the most interesting being a twofold increase in the level of a-glyeerol-P in the high response group. These observations were con~Jsten~, with a previous suggestion that severe milk fat depression can be attributed, in part, to a decrease in the availability of long-chain ~atty acids for milk synthesis. Alterations in mammary gland metabolism may occur, even though no significant enzymatic changes occur in manlma~T tissue when milk f a t percentage is depressed.
Experimental
Procedure
Eight lactating Holsteins were fed all-hay or all-concentrate rations in isocalorie amounts in a reversal trial. The all-hay ration was offered ad libitum and the concentrate ration in amounts to provide 68% of the dry matter intake of hay. Composite milk samples for determination of milk f a t percentage and f a t t y acid composition of milk fat were collected during the last week of each four-week feeding period. Samples of abdominal adipose and mammary tissues were obtained by surgical biopsy at the end of each feeding period. Tissue samples were frozen immediately after collection in liquid nitrogen and stored under liquid nitrogen until assayed. Gas-liquid chromatography of f a t t y acids and enzyme assays were done as described previously (1, 3, 14, 16). Tissue metabolite levels were determined according to methods described elsewhere (5, 10, 11, 20).
Previous studies have indicated that the milk fat depression observed when cows are fed high concentrate :low roughage diets might be due, in par~, to alterations in the metabolisms of adipose tissue, mammary tissue, or both (12, 13, 14, 19). These studies were based upon evaluations of differences in volatile f a t t y acid Received for publication August 2, 1968. 1 Preliminary reports of portions of this report were presented at the 52rid annual meeting of the Federation Association of Federated Experimental Biologists in April, 1968, and at the 63rd annual meeting of the American Dairy Science Association in June, 1968. Supported in part by U.S. Public Health Service Grant no. AM-07672. 183
Results and Discussion
Results of the enzyme studies are presented in Table 1. As observed in a previous study, no significant alterations in the activities of mammary gland enzymes were observed while very marked, three- to fourfold, changes in the activities of enzymes involved in f a t t y acid synthesis and esterification occurred in adipose tissue (4, 6, 7, 8, 10, 14, 15). The activities of glucose-6-P, 6-P-gluconate, malate ( N A D P ) , a-glycerol-P, and isoeitrate dehydrogenases were 4.3, 2.2, 3.8, 4.4, and 3.3 times higher, respectively, in adipose tissues from cows when fed concentrate as compared to hay. The activities of enzymes not usually considered related to f a t t y acid synthesis and esterification, such as
184
BALDWIN ET AL
TABLE 1. Effect of diet on activities of several enzymes in abdominal adipose and mammary tissues. Tissue and treatment Mammary Determination Glucose-6-P dehydrogenase 6-P-Gluconate dehydrogenase Ma]ate dehydrogenase (NAD) Lactate dehydrogenase Malate dehydrogenase (NADP) Hexokinase a-G]ycercI-P dehydrogenase Isocitrate dehydrogenase Protein
Adipose
Concentrate
Hay
0.268 + 0.005 ~
(~moles c o n v e r t e d / r a i n / g ) 0.305 + 0.061" 0.154 ~___0.049 a
Concentrate
Hay 0.036 + 0.016 a
1.170 ___~0.008
0.905 _____0.073
0.097 + 0.032
0.044 -+- 0.012
52.5 + 1.95 25.6 -~- 5.1
36.8 -+- 3.200 16.2 + 1.710
7.87 -+- 1.24 7.08 + 1.76
6.30 + 1.83 7.87 _____1.80
0.195 ~___0.048 0.585 _____0.007
0.206 ___~0.032 0.770 + 0.146
0.150 + 0.022 0.286 + 0'.024
0.041 + 0.007 0.202 + 0.037
0.024 ~___0.005
0.015 ± 0.005
0.012 "4- 0.012 0.053 ± 0.011
44.4 -~- 2.6
34.8 ~___2.4
0.053 _____0'.008 1.76 ~ 0.42 (mg/g) 12.5 _____3.2
11.9 ± 1.8
Standard error of mean where N ~-9 for mammary samples and N = 8 for adipose samples. hexokinase a n d malate ( N A D ) a n d lactate deh y d r o g e n a s e s , were n o t affected. The differences in the f a t t y acid compositions of milk f a t f r o m the c o n c e n t r a t e - f e d cows as c o m p a r e d to the h a y - f e d cows (Table 2 ) , although n o t so dramatic, were consistent with those p r e s e n t e d b y O p s t v e d t a n d R o n n i n g (14). D u r i n g examination o f the d a t a on diff e r e n c e s in f a t t y acid compositions o f milk f a t s f r o m different cows f e d the two diets, it was noted t h a t some individuals w e r e much nmre affected b y differences in diet t h a n were others, a n d t h a t t h e s e individual differences w e r e related to the e x t e n t o f d e p r e s s i o n of milk f a t percentage. A c o m p a r i s o n o f extent o f milk f a t d e p r e s s i o n a n d a l t e r a t i o n s in its composiTABLE 2. F a t t y acid composition of milk from cows fed hay and concentrate rations. Diet Fatty-acid ~ 6:0 8:0 10:0 12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 Milk fat, %
Concentrate
Hay
----(Area 1.6 ~_~ 0.2 0.9 + 0.1 2.8 + 0.3 3.3 -+- 0.4 9.7 -+- 0.5 1.9 + 0.3 24.7 -+- 0.6 3.3 + 0.2 11.1 + 1.1 29.1 ~___1.5 5.7 _____0.6 1.1 + 0.1 2.2 + 0.1
% ) 2.1 1.0 2.8 3.1 9.7 2.3 26.6 3.9 11.2 27.9 2.9 1.9 3.4
- _____0.2 ± 0.1 _____0.3 + 0.4 + 0.6 + 0.2 + 0.6 + 0.1 _____0.6 _____2.1 -+- 0.3 _____0.2 + 0.1
Expressed as carbon number :number of double bonds. Values presented represent means and standard errors of the means of percentage of total f a t t y acid based on peak area. J. DAIRYSCIENCE V O L
52, NO,. 2
tion in t h r e e cows (low r e s p o n s e g r o u p ) whose milk f a t p e r c e n t a g e s were depressed only slightly ( 0 . 6 % ) , to t h r e e cows (high r e s p o n s e g r o u p ) whose milk f a t p e r c e n t a g e s were dep r e s s e d more m a r k e d l y (1.8%) b y the concent r a t e diet, is p r e s e n t e d in Table 3. The differences in f a t t y acid content o f milk f a t f r o m cows in the high r e s p o n s e g r o u p were consist e n t with t h e results o f O p s t v e d t a n d R o n n i n g (14), whereas the changes observed in the low r e s p o n s e g r o u p were not (Table 3). The diff e r e n c e s in f a t t y acid composition of milk f a t in the h i g h and low r e s p o n s e g r o u p s were significantly different ( P ~ 0.05). On the basis o f this observation, it was decided t h a t comp a r i s o n o f metabolic d a t a obtained with animals in t h e high and low r e s p o n s e g r o u p s m i g h t yield i n s i g h t s relative to metabolic causes of depressions in milk f a t p e r c e n t a g e a n d o f differences in individual responses. I t was also believed t h a t such a c o m p a r i s o n would eliminate the difficulty of d e t e r m i n i n g w h e t h e r alteraTABLE 3. Differences in milk fat, 18:0, and 18:1 between animals in high and low response groups. ~ Response group Milk fat Low High
(%) --0.6 ~ 0.12 --1.8 ~ 0.11
Change in 18 :Ob
18:1 b
~ - (Area % ) - +2.6 _____0.91 --1.5 ----+-0.75 --3.3 + 0.88 +5.2 + 1.51
a Values represent mean differences between milk compositions when cows were fed hay as compared to concentrate rations (hay values-concentrate values) ~ standard errors of mean differences with N = 4. b F a t t y acids expressed as carbon number:number of double bonds.
MAMMARY AND ADIPOSE TISSUE tions in enzymatic activities of adipose tissue reflected merely a diet effect or a cause-andeffect relationship in the depression of milk fat percentage. Comparison of enzymatic data between the high and low response groups was made difficult by the small number of individuals represented in each group; however, two notable differences were evident. First, it was noted that some enzymatic activities in the adipose tissues of cows in the low response group were lower than in the high response group. For example, on the concentrate diet, the activities of glucose-6-P, 6-P-glueonate, and aglycerol-P dehydrogenases were 0.06, 0.06, and 0.02 unit × ]0:/milligram protein, respectively, in the low response group, and 0.40, 0.53, and 0.09 unit × 10"/milligram protein, respectively, in the high response group. The second notable difference was an apparent tendency for greater enzyme adaptations in adipose samples from cows in the high response group. One of the primary difficulties in the interpretation of enzymatic data is that changes in enzymatic activities, although usually indicative of metabolic changes, are not necessarily accompanied by a metabolic change. Conversely, a change in metabolic activity can occur in the absence of changes in enzyme activities. The reason for potential lack of correspondence between enzyme activities is that shifts in tissue levels of enzyme substrates and products can occur which compensate for changes in enzyme activities or produce metabolic changes in the absence of enzymatic adaptations. Therefore, a limited study o£ tissue levels of a number of intermediate metabolites was conducted to determine whether enzymatic
185
changes were accompanied by metabolic changes. The levels of metabolites observed in mammary and adipose tissues of cows fed hay and concentrate diets are presented in Table 4. Due to the quantities of tissue required for analysis of tissue metabolites and the small size of our biopsy samples, it was necessary to pool samples from several animals for each analysis. Since it had been observed that individual differences in degree of milk fat depression were related to differences in milk fatty acid compositions on the two diets and, also, to enzyme differences, it was decided to pool samples according to the high and low response groups defined above. The values in Table 4 represent the results from an analysis of a pooled sample of four or five samples. The samples were obtained from three different individuals in eacb response group. Previous experiences in interpretation of metabolite data where it was possible to assess variance indicated that the differences of 50-80% are required to obtain statistical significance with this number of samples, with the exception that the ratio of ADP + 2 A T P to 2(AMP -~ ADP + A T P ) is very consistent and differences of 10-15% are normally statistically significant with four or five estimates per group (2). Several of the differences in nlammary gland metabolite levels (Table 4) seem noteworthy. Levels of phosphoenol-pyruvate ( P E P ) were five to ten times higher in both groups when they were fed concentrate as compared to hay, possibly indicating a limitation in pyruvate kinase activity. Malate levels appeared to be depressed and a-glycerol-P and A T P levels
TABLE 4. Metabolite patterns in abdominal adipose and mammary tissues of cows in high and low response groups. Mammary Metabolite Glucose Glucose-6-P Fructose-l,6-diP Triose-P a-GlyeeroLP Phosphoenolpyruvate Pyruvate Lactate Malate a-Ketog]utarate Glutamate ADP ~- 2ATP
High
Adipose
Concentrate
Hay Low
High
Low
(m~ moles/g) 2,081 607 76 11 7.4 1.0 139 3.2 264 188 11.7 15 28 25 1,114 562 0.8 12 27 10 3,227 498
1,891 46 4.9 222 154 0.5 49 1,509 7.3 31 2,970
1,812 36 7.4 192 79 2.6 22 1,633 6.1 21 2,618
1,482 46 8.4 170 205 4.1 17 727 2.9 14 2,654
0.53
0.42
0.67
0.77
Concentrate
Hay High
0.46
Low
High
Low
621 7 0.9 4.2 162 16 26 540 22 10 498
820 29 0.8 6.8 331 21 26 647 35 13 1,523
1,007 18 1.2 3.8 149 18 29 706 53 17 1,358
0.48
0.69
0.62
2 ( A M P + A D P + ATP) J. I)AII~Y SCIENCE VOL. 52, NO. 2
186
B A L D W I N E T AL
increased by concentrate feeding. I t seems notable that the levels of glucose, glucose-6-P, P E P , pyruvate, lactate, and a-ketofflutarate were all lower in the high response cows fed concentrate than in the low response cows fed concentrate. A n attractive interpretation of this observation is that it reflects a decreased energy availability to the mammary glands of cows in the high response groups as compared to the animals in the low response group. There are a number of other explanations, however, which are approximately equally consistent with the limited data available. The most important conclusion that can be drawn from consideration of mammaxT metabolite levels is that although no enzymatic adaptations to high-concentrate diets are observed in the mammary gland, metabolic changes may occur which could be related to milk secretion. There appear to be a number of metabolite changes which occur in adipose tissue when high-concentrate diets are fed (Table 4). Tissue levels of glucose, glucose-6-P, malate, and glutamate, and the ratio of ADP + 2 A T P / 2(AiV[P + ADP + A T P ) were all increased markedly, most probably indicating an increased availability of substrates to adipose tissue in concentrate-fed animals. When this suggestion is considered along with the observed increases in enzymatic capacity in adipose tissues of concentrate-fed animals (Table 1) and the fact that the increases in enzymatic capacity are restricted to enzymes whose activities are related to fatty acid synthesis and fatty acid esterification, it does not seem inconsistent to conclude that high-concentrate diets cause an increase in the storage activity o1" function of adipose tissue in lactating cows. This conclusion is consistent with observations and conclusions from a number of workers (4, 9, 12, 13, 14, 17). Only two significant differences in metabolite levels in adipose tissues between cows in the high and low response groups were evident. The levels of triose-P and a-glycerol-P were higher in the high response group. The higher level of triose-P, occurring as it does in the absence of an increase in fructose-l,6-diP, could reflect an increase in rate of formation via the hexose monophosphate pathway. The difference in a-glycerol-P levels is especially notable, since it was proposed previously (13) that the rate of fatty acid esterification is elevated in cows whose milk fat production is depressed; that this increased rate of fatty acid esterification resulted in a decrease in the availability of milk fat precursors to the mammary gland. This suggestion was supported by observations that g. DAIRY SCIENCE VO~. 52, NO. 2
the proportions of fatty acids derived from adipose tissue were depressed in cows with depressed milk fat production and that adipose a-glycerol-P dehydrogenase activity was elevated in cows fed milk fat-depressing diets (8, 13, 14). I n our study these two observations were confirmed (Tables 1 and 3) and the predicted elevation in a-glycerol-P level required for increased rates of fatty acid esterification in adipose tissue was observed in cows with severely depressed milk fat percentages (Table 4). The fact that elevated adipose a-glycerolP levels were observed only in cows in the high response group lends credence to the postulate, in that the difference can be related to milk fat depression. Although the separation of the cows studied into high and low response groups can be considered arbitrary, in the sense that no statistical validation of the separation is feasible with the limited number of individuals involved, it is suggested on the basis of the data presented that changes in diet produce changes in enzyme and metabolite patterns not related to degree of milk fat depression and that, hence, separation of animals into response groups might be more than, or as appropriate as, simple comparisons between diets without consideration of physiological responses. References
(1) Baldwin, R. L. 1966. Emzymatic activities in mammary glands of several species. J. Dairy Sci., 49: 1533. (2) Baldwin, R. L., and W. Cheng. 1968. Metabolite changes associated with the initiation and maintenance of lactation in rats and cows. Unpublished data. (3) Ba]dwin, R. L., and L. P. Milligan. 1966. Enzymatic changes associated with the initiation and maintenance of lactation in the rat. J. Biol. Chem., 241: 2058. (4) Barry, J. M., W. Bartley, J. L. Linzell, and D. S. Robinson. 1963. The uptake from the blood of triglyceride fatty acids of chylomiera and low-density lipoproteins by the mammaxy gland of the goat. Biochem. J., 89: 6. (5) Bergmeyer, H. U. 1963. Methods of Enzymatic Analysis. Academic Press, New York. (6) Flatt, J. P., and E. G. Ball. 1966. Studies on the metabolism of adipose tissue. X I X . An evaluation of the major pathways of glucose catabolism as influenced by acetate in the presence of insulin, g. Biol. Chem., 241 : 2862. (7) Hibbitt, K. G. 1966. Some factors involved in the control of fatty acid synthesis in the lactating bovine mammary gland. Biochim. et Biophys. Aeta, 116 : 56.
MAMMARY AND ADIPOSE TISSUE (8) Howard, C. F., Jr., and J. M. Lowenstein. 1965. The effect of glycerol 3-phosphate on f a t t y acid synthesis. J. Biol. Chem., 2 4 0 : 4170. (9) Jorgensen, N. A., L. H. Schultz, and G. R. Earr. 1965. 1%~tors influencing milk f a t depression on rations high in concentrates. J. Dairy Sci., 48: 1031. (10) Lowry, O. H., J. V. Passoneau, F. X. Hasselberger, and D. W. Sehultz. 1964. Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. J. Biol. Chem., 239: 18. (11) Maitra, P. K., and R. W. Estabrook. 1964. A fluorometric method for the enzymic determination of glycolytic intermediates. Anal. Biochem., 7:472. (12) l~cClymont, G. L., and S. Vallanee. 1962. Depression of blood glycerides and milkf a t synthesis by glucose infusion. Proe. Nutrition Soc., 2 1 : X I I . (13) Opstvedt, J., R. L. Baldwin, and M. Ronning. 1967. Effect of diet upon activities of several enzymes in abdominal adipose and mammary tissues in the laatating dairy cow. J. Dairy Sei., 50: 108. (14) Opstvedt, J., and M. Ronning. 1967. Effect upon lipid metabolism of feeding alfalfa hay or concentrate ad libitum as the sole
(15)
(16)
(17)
(18)
(19)
(20)
187
feed for milking cows. J. Dairy Sci., 50: 345. Shaw, J. C., R. R. Robinson, M. E. Senger, S. Lakstnna~an, and T. R. Lewis. 1959. Production of low-fat milk. I. Effect of quality and quantity of concentrate on tureen volatile f a t t y acids and milk composition. J. Nutrition, 69: 235. Smith, L. M. 1961. Quantitative f a t t y acid analysis of milk f a t by gas-llquid chromatography. J . Dairy Sci., 44: 607. Sterry, J. E., a~d J. A. F. Rook. 1965. The effects of a diet low in hay and high in flaked maize on milk f a t secretion and on the concentration of certain constituents in the blood plasma of the cow. Brit. J. Nutrition, 19: 101. Tepperman, J., and H. M. Tepperman. 1965. Adaptive hyperlipogenesis--late, 1964, model. Ann. N.Y. Acad. Sci., 131: 404. Van Soest, P. J. 1963. Ruminant f a t metabolism with particular reference to factors affecting low milk f a t and feed efficiency. A review. J. Dairy Sci., 46:204. Williamson, J. R. 1965. Glycolytic control mechanisms. I. Inhibition of glycolysis by acetate and pyruvate in the isolated, perfused rat heart. J. Biol. Chem., 240: 2308.
J. DAIRY SCIENCE VOL. 52, NO. 2
Department of Animal Science, University of California, Davis Abstract
production in the rumen (15, 18, 19), of changes in the patterns of f a t t y acids in milk f a t (14, 19), of alterations in blood metabolites (17), and of indications of metabolic adaptations in adipose and mammary tissue (13, 19). Our study was undertaken to confirm a previous observation that marked changes in the levels of several enzymes occur in adipose tissues of cows fed high concentrate diets (13, 14) and to attempt to ascertain the metabolic significance of these changes.
Compared to hay, when concentrates were fed three- to fourfold, increases occurred in the activities of enzymes associated with f a t t y acid synthesis and esterification in adipose tissue, whereas mammary enzyme levels were relatively unaffected. The proportion of stearie acid in milk fat, presumably arising from adipose tissue, was significantly depressed in the high response group (severe milk fat depression) but not in the low response group (moderate milk f a t depression). Differences in dietary effects upon mammary intermediary metabolite levels between the high and low response groups suggested a relatively lower availability of energy to mammary glands of cows in the high response group when fed concentrate. A number of changes in adipose tissue metabolite levels were noted in concentratefed animals, the most interesting being a twofold increase in the level of a-glyeerol-P in the high response group. These observations were con~Jsten~, with a previous suggestion that severe milk fat depression can be attributed, in part, to a decrease in the availability of long-chain ~atty acids for milk synthesis. Alterations in mammary gland metabolism may occur, even though no significant enzymatic changes occur in manlma~T tissue when milk f a t percentage is depressed.
Experimental
Procedure
Eight lactating Holsteins were fed all-hay or all-concentrate rations in isocalorie amounts in a reversal trial. The all-hay ration was offered ad libitum and the concentrate ration in amounts to provide 68% of the dry matter intake of hay. Composite milk samples for determination of milk f a t percentage and f a t t y acid composition of milk fat were collected during the last week of each four-week feeding period. Samples of abdominal adipose and mammary tissues were obtained by surgical biopsy at the end of each feeding period. Tissue samples were frozen immediately after collection in liquid nitrogen and stored under liquid nitrogen until assayed. Gas-liquid chromatography of f a t t y acids and enzyme assays were done as described previously (1, 3, 14, 16). Tissue metabolite levels were determined according to methods described elsewhere (5, 10, 11, 20).
Previous studies have indicated that the milk fat depression observed when cows are fed high concentrate :low roughage diets might be due, in par~, to alterations in the metabolisms of adipose tissue, mammary tissue, or both (12, 13, 14, 19). These studies were based upon evaluations of differences in volatile f a t t y acid Received for publication August 2, 1968. 1 Preliminary reports of portions of this report were presented at the 52rid annual meeting of the Federation Association of Federated Experimental Biologists in April, 1968, and at the 63rd annual meeting of the American Dairy Science Association in June, 1968. Supported in part by U.S. Public Health Service Grant no. AM-07672. 183
Results and Discussion
Results of the enzyme studies are presented in Table 1. As observed in a previous study, no significant alterations in the activities of mammary gland enzymes were observed while very marked, three- to fourfold, changes in the activities of enzymes involved in f a t t y acid synthesis and esterification occurred in adipose tissue (4, 6, 7, 8, 10, 14, 15). The activities of glucose-6-P, 6-P-gluconate, malate ( N A D P ) , a-glycerol-P, and isoeitrate dehydrogenases were 4.3, 2.2, 3.8, 4.4, and 3.3 times higher, respectively, in adipose tissues from cows when fed concentrate as compared to hay. The activities of enzymes not usually considered related to f a t t y acid synthesis and esterification, such as
184
BALDWIN ET AL
TABLE 1. Effect of diet on activities of several enzymes in abdominal adipose and mammary tissues. Tissue and treatment Mammary Determination Glucose-6-P dehydrogenase 6-P-Gluconate dehydrogenase Ma]ate dehydrogenase (NAD) Lactate dehydrogenase Malate dehydrogenase (NADP) Hexokinase a-G]ycercI-P dehydrogenase Isocitrate dehydrogenase Protein
Adipose
Concentrate
Hay
0.268 + 0.005 ~
(~moles c o n v e r t e d / r a i n / g ) 0.305 + 0.061" 0.154 ~___0.049 a
Concentrate
Hay 0.036 + 0.016 a
1.170 ___~0.008
0.905 _____0.073
0.097 + 0.032
0.044 -+- 0.012
52.5 + 1.95 25.6 -~- 5.1
36.8 -+- 3.200 16.2 + 1.710
7.87 -+- 1.24 7.08 + 1.76
6.30 + 1.83 7.87 _____1.80
0.195 ~___0.048 0.585 _____0.007
0.206 ___~0.032 0.770 + 0.146
0.150 + 0.022 0.286 + 0'.024
0.041 + 0.007 0.202 + 0.037
0.024 ~___0.005
0.015 ± 0.005
0.012 "4- 0.012 0.053 ± 0.011
44.4 -~- 2.6
34.8 ~___2.4
0.053 _____0'.008 1.76 ~ 0.42 (mg/g) 12.5 _____3.2
11.9 ± 1.8
Standard error of mean where N ~-9 for mammary samples and N = 8 for adipose samples. hexokinase a n d malate ( N A D ) a n d lactate deh y d r o g e n a s e s , were n o t affected. The differences in the f a t t y acid compositions of milk f a t f r o m the c o n c e n t r a t e - f e d cows as c o m p a r e d to the h a y - f e d cows (Table 2 ) , although n o t so dramatic, were consistent with those p r e s e n t e d b y O p s t v e d t a n d R o n n i n g (14). D u r i n g examination o f the d a t a on diff e r e n c e s in f a t t y acid compositions o f milk f a t s f r o m different cows f e d the two diets, it was noted t h a t some individuals w e r e much nmre affected b y differences in diet t h a n were others, a n d t h a t t h e s e individual differences w e r e related to the e x t e n t o f d e p r e s s i o n of milk f a t percentage. A c o m p a r i s o n o f extent o f milk f a t d e p r e s s i o n a n d a l t e r a t i o n s in its composiTABLE 2. F a t t y acid composition of milk from cows fed hay and concentrate rations. Diet Fatty-acid ~ 6:0 8:0 10:0 12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 Milk fat, %
Concentrate
Hay
----(Area 1.6 ~_~ 0.2 0.9 + 0.1 2.8 + 0.3 3.3 -+- 0.4 9.7 -+- 0.5 1.9 + 0.3 24.7 -+- 0.6 3.3 + 0.2 11.1 + 1.1 29.1 ~___1.5 5.7 _____0.6 1.1 + 0.1 2.2 + 0.1
% ) 2.1 1.0 2.8 3.1 9.7 2.3 26.6 3.9 11.2 27.9 2.9 1.9 3.4
- _____0.2 ± 0.1 _____0.3 + 0.4 + 0.6 + 0.2 + 0.6 + 0.1 _____0.6 _____2.1 -+- 0.3 _____0.2 + 0.1
Expressed as carbon number :number of double bonds. Values presented represent means and standard errors of the means of percentage of total f a t t y acid based on peak area. J. DAIRYSCIENCE V O L
52, NO,. 2
tion in t h r e e cows (low r e s p o n s e g r o u p ) whose milk f a t p e r c e n t a g e s were depressed only slightly ( 0 . 6 % ) , to t h r e e cows (high r e s p o n s e g r o u p ) whose milk f a t p e r c e n t a g e s were dep r e s s e d more m a r k e d l y (1.8%) b y the concent r a t e diet, is p r e s e n t e d in Table 3. The differences in f a t t y acid content o f milk f a t f r o m cows in the high r e s p o n s e g r o u p were consist e n t with t h e results o f O p s t v e d t a n d R o n n i n g (14), whereas the changes observed in the low r e s p o n s e g r o u p were not (Table 3). The diff e r e n c e s in f a t t y acid composition of milk f a t in the h i g h and low r e s p o n s e g r o u p s were significantly different ( P ~ 0.05). On the basis o f this observation, it was decided t h a t comp a r i s o n o f metabolic d a t a obtained with animals in t h e high and low r e s p o n s e g r o u p s m i g h t yield i n s i g h t s relative to metabolic causes of depressions in milk f a t p e r c e n t a g e a n d o f differences in individual responses. I t was also believed t h a t such a c o m p a r i s o n would eliminate the difficulty of d e t e r m i n i n g w h e t h e r alteraTABLE 3. Differences in milk fat, 18:0, and 18:1 between animals in high and low response groups. ~ Response group Milk fat Low High
(%) --0.6 ~ 0.12 --1.8 ~ 0.11
Change in 18 :Ob
18:1 b
~ - (Area % ) - +2.6 _____0.91 --1.5 ----+-0.75 --3.3 + 0.88 +5.2 + 1.51
a Values represent mean differences between milk compositions when cows were fed hay as compared to concentrate rations (hay values-concentrate values) ~ standard errors of mean differences with N = 4. b F a t t y acids expressed as carbon number:number of double bonds.
MAMMARY AND ADIPOSE TISSUE tions in enzymatic activities of adipose tissue reflected merely a diet effect or a cause-andeffect relationship in the depression of milk fat percentage. Comparison of enzymatic data between the high and low response groups was made difficult by the small number of individuals represented in each group; however, two notable differences were evident. First, it was noted that some enzymatic activities in the adipose tissues of cows in the low response group were lower than in the high response group. For example, on the concentrate diet, the activities of glucose-6-P, 6-P-glueonate, and aglycerol-P dehydrogenases were 0.06, 0.06, and 0.02 unit × ]0:/milligram protein, respectively, in the low response group, and 0.40, 0.53, and 0.09 unit × 10"/milligram protein, respectively, in the high response group. The second notable difference was an apparent tendency for greater enzyme adaptations in adipose samples from cows in the high response group. One of the primary difficulties in the interpretation of enzymatic data is that changes in enzymatic activities, although usually indicative of metabolic changes, are not necessarily accompanied by a metabolic change. Conversely, a change in metabolic activity can occur in the absence of changes in enzyme activities. The reason for potential lack of correspondence between enzyme activities is that shifts in tissue levels of enzyme substrates and products can occur which compensate for changes in enzyme activities or produce metabolic changes in the absence of enzymatic adaptations. Therefore, a limited study o£ tissue levels of a number of intermediate metabolites was conducted to determine whether enzymatic
185
changes were accompanied by metabolic changes. The levels of metabolites observed in mammary and adipose tissues of cows fed hay and concentrate diets are presented in Table 4. Due to the quantities of tissue required for analysis of tissue metabolites and the small size of our biopsy samples, it was necessary to pool samples from several animals for each analysis. Since it had been observed that individual differences in degree of milk fat depression were related to differences in milk fatty acid compositions on the two diets and, also, to enzyme differences, it was decided to pool samples according to the high and low response groups defined above. The values in Table 4 represent the results from an analysis of a pooled sample of four or five samples. The samples were obtained from three different individuals in eacb response group. Previous experiences in interpretation of metabolite data where it was possible to assess variance indicated that the differences of 50-80% are required to obtain statistical significance with this number of samples, with the exception that the ratio of ADP + 2 A T P to 2(AMP -~ ADP + A T P ) is very consistent and differences of 10-15% are normally statistically significant with four or five estimates per group (2). Several of the differences in nlammary gland metabolite levels (Table 4) seem noteworthy. Levels of phosphoenol-pyruvate ( P E P ) were five to ten times higher in both groups when they were fed concentrate as compared to hay, possibly indicating a limitation in pyruvate kinase activity. Malate levels appeared to be depressed and a-glycerol-P and A T P levels
TABLE 4. Metabolite patterns in abdominal adipose and mammary tissues of cows in high and low response groups. Mammary Metabolite Glucose Glucose-6-P Fructose-l,6-diP Triose-P a-GlyeeroLP Phosphoenolpyruvate Pyruvate Lactate Malate a-Ketog]utarate Glutamate ADP ~- 2ATP
High
Adipose
Concentrate
Hay Low
High
Low
(m~ moles/g) 2,081 607 76 11 7.4 1.0 139 3.2 264 188 11.7 15 28 25 1,114 562 0.8 12 27 10 3,227 498
1,891 46 4.9 222 154 0.5 49 1,509 7.3 31 2,970
1,812 36 7.4 192 79 2.6 22 1,633 6.1 21 2,618
1,482 46 8.4 170 205 4.1 17 727 2.9 14 2,654
0.53
0.42
0.67
0.77
Concentrate
Hay High
0.46
Low
High
Low
621 7 0.9 4.2 162 16 26 540 22 10 498
820 29 0.8 6.8 331 21 26 647 35 13 1,523
1,007 18 1.2 3.8 149 18 29 706 53 17 1,358
0.48
0.69
0.62
2 ( A M P + A D P + ATP) J. I)AII~Y SCIENCE VOL. 52, NO. 2
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increased by concentrate feeding. I t seems notable that the levels of glucose, glucose-6-P, P E P , pyruvate, lactate, and a-ketofflutarate were all lower in the high response cows fed concentrate than in the low response cows fed concentrate. A n attractive interpretation of this observation is that it reflects a decreased energy availability to the mammary glands of cows in the high response groups as compared to the animals in the low response group. There are a number of other explanations, however, which are approximately equally consistent with the limited data available. The most important conclusion that can be drawn from consideration of mammaxT metabolite levels is that although no enzymatic adaptations to high-concentrate diets are observed in the mammary gland, metabolic changes may occur which could be related to milk secretion. There appear to be a number of metabolite changes which occur in adipose tissue when high-concentrate diets are fed (Table 4). Tissue levels of glucose, glucose-6-P, malate, and glutamate, and the ratio of ADP + 2 A T P / 2(AiV[P + ADP + A T P ) were all increased markedly, most probably indicating an increased availability of substrates to adipose tissue in concentrate-fed animals. When this suggestion is considered along with the observed increases in enzymatic capacity in adipose tissues of concentrate-fed animals (Table 1) and the fact that the increases in enzymatic capacity are restricted to enzymes whose activities are related to fatty acid synthesis and fatty acid esterification, it does not seem inconsistent to conclude that high-concentrate diets cause an increase in the storage activity o1" function of adipose tissue in lactating cows. This conclusion is consistent with observations and conclusions from a number of workers (4, 9, 12, 13, 14, 17). Only two significant differences in metabolite levels in adipose tissues between cows in the high and low response groups were evident. The levels of triose-P and a-glycerol-P were higher in the high response group. The higher level of triose-P, occurring as it does in the absence of an increase in fructose-l,6-diP, could reflect an increase in rate of formation via the hexose monophosphate pathway. The difference in a-glycerol-P levels is especially notable, since it was proposed previously (13) that the rate of fatty acid esterification is elevated in cows whose milk fat production is depressed; that this increased rate of fatty acid esterification resulted in a decrease in the availability of milk fat precursors to the mammary gland. This suggestion was supported by observations that g. DAIRY SCIENCE VO~. 52, NO. 2
the proportions of fatty acids derived from adipose tissue were depressed in cows with depressed milk fat production and that adipose a-glycerol-P dehydrogenase activity was elevated in cows fed milk fat-depressing diets (8, 13, 14). I n our study these two observations were confirmed (Tables 1 and 3) and the predicted elevation in a-glycerol-P level required for increased rates of fatty acid esterification in adipose tissue was observed in cows with severely depressed milk fat percentages (Table 4). The fact that elevated adipose a-glycerolP levels were observed only in cows in the high response group lends credence to the postulate, in that the difference can be related to milk fat depression. Although the separation of the cows studied into high and low response groups can be considered arbitrary, in the sense that no statistical validation of the separation is feasible with the limited number of individuals involved, it is suggested on the basis of the data presented that changes in diet produce changes in enzyme and metabolite patterns not related to degree of milk fat depression and that, hence, separation of animals into response groups might be more than, or as appropriate as, simple comparisons between diets without consideration of physiological responses. References
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MAMMARY AND ADIPOSE TISSUE (8) Howard, C. F., Jr., and J. M. Lowenstein. 1965. The effect of glycerol 3-phosphate on f a t t y acid synthesis. J. Biol. Chem., 2 4 0 : 4170. (9) Jorgensen, N. A., L. H. Schultz, and G. R. Earr. 1965. 1%~tors influencing milk f a t depression on rations high in concentrates. J. Dairy Sci., 48: 1031. (10) Lowry, O. H., J. V. Passoneau, F. X. Hasselberger, and D. W. Sehultz. 1964. Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. J. Biol. Chem., 239: 18. (11) Maitra, P. K., and R. W. Estabrook. 1964. A fluorometric method for the enzymic determination of glycolytic intermediates. Anal. Biochem., 7:472. (12) l~cClymont, G. L., and S. Vallanee. 1962. Depression of blood glycerides and milkf a t synthesis by glucose infusion. Proe. Nutrition Soc., 2 1 : X I I . (13) Opstvedt, J., R. L. Baldwin, and M. Ronning. 1967. Effect of diet upon activities of several enzymes in abdominal adipose and mammary tissues in the laatating dairy cow. J. Dairy Sei., 50: 108. (14) Opstvedt, J., and M. Ronning. 1967. Effect upon lipid metabolism of feeding alfalfa hay or concentrate ad libitum as the sole
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