- Email: [email protected]
The effect of initiated involution on enzyme activity and substrate uptake by the mammary gland of the lactating rabbit
Iur. J Printed
Biochrnl. in Great
Vol.
IS, No. 4, pp. 565-569.
Britain.
All rights
1983
0020.7
reserved
CopyrIght
0
I IXi83:040565-05$03.00/O 1983 Pergamon
Press
Ltd
THE EFFECT OF INITIATED INVOLUTION ON ENZYME ACTIVITY AND SUBSTRATE UPTAKE BY THE MAMMARY GLAND OF THE LACTATING RABBIT COLIN S. JONES* and DAVID S. PARKERt Department of Physiology and Biochemistry, University of Reading, Whiteknights, Reading RG6 2AJ, U.K. 13 July 1982)
(Received
Arteriovenous difference studies across the lactating rabbit mammary gland for glucose, acetate, triacylglycerol and non-esterified fatty acids during initiated involution are reported. 2. A significant reduction in substrate utilisation is paralleled by a decrease in the activities of fatty acid synthetase, acetyl CoA synthetase, citrate synthase and glutamate dehydrogenase in biopsy samples
Abstract--l.
taken from the gland. 3. Results from the analysis
of linid fractions
within
the gland
during
this period
are discussed
in
relation to lipid resorption.
INTRODUCTION
aseptically from a separate group of does. A small incision was made in the skin covering one of the abdominal glands and tissue excised using diathermy. The tissue was homogenised in 0.25 M sucrose and fractionated into sub-cellular components by the method described by Ash & Baird (1973). Fractions were stored at -20” prior to assay for specific enzymes. All assay procedures were checked for linearity with time and protein concentration; the individual assay systems used were: glutamate dehydrogenase (Schmidt, 1974); fatty acid synthetase (Martin et al., 1961); citrate synthase (Srere et al., 1963); acetyl CoA synthetase (Huang, 1970) and lactate dehydrogenase (Bergmeyer & Brent, 1974). Tissue lipids were separated into individual components by t.1.c. and the TAG and NEFA fractions prepared for g.1.c. (Jones & Parker, 1978).
Mammary gland arteriovenous difference studies and blood flow measurements in the lactating rabbit have demonstrated the importance of various metabolites in the metabolism of the gland and as precursors for milk synthesis (Jones & Parker, 1978; Jones & Parker, 1981). During initiated involution of the mammary gland brought about by the premature removal of the young, there has been shown to be a rapid reduction in metabolic activity within the gland of the rat (Jones, 1967, 1968) and the ewe (Bauman et al., 1974). This is followed by regression of the mammary tissue and resorption of the milk constituents (see review by Lascelles & Lee, 1978). The following experiments were designed to investigate the response of the lactating rabbit mammary gland to initiated involution with reference to metabolite uptake by the gland and also tissue enzyme studies and lipid content measured in biopsy samples.
RESULTS AND DlSCUSSlON The activities of four of the enzymes studied show a significant fall immediately after the removal of the young. This is reflected in both the mitochondrial glutamate dehydrogenase and citrate synthase activities and the cytosolic enzymes fatty acid synthetase and acetyl CoA synthetase (Fig. 1). The activity of lactate
METHODS Lactating New Zealand White rabbits (Buxted Rabbits, Buxted, Sussex) were obtained at around day 14 of lactation and housed under conditions of 16 hr day length.
dehydrogenase (33.0 + 2.6 (21) pmol substrate converted/min per g wet wt tissue) did not change during the period studied. These results indicate a rapid response within the tissue to the cessation of milk removal and are in agreement with data for other enzymes in the rat (Jones, 1967, 1968), the sheep (Bauman et ul., 1974) and one other study in the rabbit (GUI & Dils, 1969). In addition to changes in the activity it is also of interest that the intracellular distribution of both the mitochondrial enzymes changed during the course of the period studied, with increased activity being detected in the cytosolic fraction as involution progressed. This may indicate increased leakage of enzymes from mitochondria as cell lysis progresses during involution. Histological examination of tissue taken from rabbits during this study show the presence of macrophages and cytosegresomes as has been reported in sheep and rat involuting tissue (Lascelles & Lee, 1978).
Food (Diet 18, Dixon & Sons, Ware, Herts) and water were available ad lihitum. The day that the young were removed from the doe was designated Day 0 of involution. The carotid artery and both lateral thoracic veins were cannulated as previously described (Jones & Parker, 1978). Blood sampling commenced within 48 hr and samples were obtained once a day for as long as the cannulae remained open. Blood samples were analysed for glucose, acetate, triacylglycerol (TAG) and non-esterified fatty acids (NEFA) as previously described (Jones & Parker, 1978). Biopsy samples of mammary tissue (500 mg) were obtained
* Present address: Department of Zoology, University of Durham, Durham. t Present address: Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, Newcastle upon Tyne. 565
COLIN S. JONES and DAVIU
S. PARKER
Acetyl CoA synthetose 6.0/-
--
2
4
6 Days after
8
Fatty
of the
synthetase
4
2
IO
removal
acid
6
IO
young
Fig. 1. Activity of rabbit mammary gland enzymes assayed in biopsy samples taken during invoiution. Activity expressed as pmol substrate converted/min per g wet wt of tissue, mean f SE. In all cases enzyme activities determined from Day 3 to Day 10 were significantly different (P c: 0.1) from that determined on Day 0. Number of biopsy samples; Day 0 = 4, Day 2 = 4, Day 3 = 4. Day 6 = 3. Day 8 = 3, Day 10 = 3.
Arteriovenous difference studies for the mammary gland underline the changes in metabolic activity occurring after removal of the pups. The results for glucose, acetate, TAG and NEFA are shown in Fig. 2. Uptake of both glucose and acetate falls rapidly within 48 hr of the removal of the young and from then until 10 days of involution remains almost zero. Both glucose and acetate are utilised extensively in the lactating rabbit mammary gland for lipid synthesis and oxidative pathways (Crabtree et al., 1981). Studies using labelled substrate indicate that these two metabolites account for SO”/, of total CO2 output by the lactating gland (Jones & Parker, 1981) and the
reduction in uptake during involution reflects the fall in metabolic activity within the gland at this time. The arteriovenous difference values for TAG also show a reduction in uptake by the gland in the first 2 days of involution although the data for some of the later samples shows an apparent reversal of this trend. These mean values have large standard errors associated with them due to some individual rabbits having very high plasma TAG values. The data for NEFA are, however, much clearer and show a very interesting pattern. The uptake of NEFA by the lactating gland noted in a previous study (Jones & Parker, 1978) changes to a net output of NEFA by the gland
Involution
of rabbit
mammary
gland
561
I2H E
IO08
-
i 8
06-
1 3
04
-
0.2
-
Uptake Output
L
t
0
2 Days
4 after
6 removal
8 of
the
IO young
Fig. 2. Arteriovenous difference values for glucose, acetate, TAG and NEFA across the rabbit mammary gland during involution. Mean LSE, dotted line indicates no net uptake or output by the mammary gland. Number of individual samples analysed were as follows; Day 0 = 10, Days 1, 2 = 2, Day 3 = 7, Day 4 = 8, Days 5, 6 = 6, Days 7, 8 = 5, Day 9 = 4, Day 10 = 3.
48 hr after removal of the young. This reaches a maximum at about 72 hr and then remains constant for the remainder of the period studied. The results of analysis of the individual fatty acids within the NEFA fraction (Fig. 3) show that this increase is primarily in the C16:0, C18:O and C18: 1 fatty acids and that the medium chain length fatty acids C8:O and ClO:O characteristic of rabbit milk are not released into the venous blood. NEFA present in mammary venous blood during lactation are derived from both the arterial supply to the gland and also from fatty acids released from plasma TAG following the action of lipoprotein lipase on the endothelial cell surface
(Moore & Christie, 1981). During involution, however, the activity of this enzyme has been shown to decline rapidly in both guinea-pig (McBride & Korn, 1963) and rat (Hamosh et al., 1970) mammary glands following removal of the young. The positive arteriovenous differences reported in the present study may, therefore, reflect loss of fatty acids from the cells of the involuting rabbit mammary gland. Tissue lipid extracted from biopsy samples during the same period were separated into NEFA and TAG fractions and the individual fatty acids from each fraction are shown in Table 1. The composition of the NEFA fraction did not alter during the 10 days stud-
COLIN S. JONESand DAVID S. PARKER
568
RUptake
Days
after
removal
of
the
young
Fig. 3. Arteriovenous difference values (PM) for individual plasma NEFA across the rabbit mammary gland during involution. Mean + SE, dotted line indicates no net uptake or output by the mammary gland. Number of individual samples analysed were as follows; Day 0 = 10, Days 1, 2 -i 2, Day 3 = 7, Day 4 = 8. Days 5, 6 = 5, Day 9 = 4, Day 10 = 3.
Table
1. Molar
proportions
of fatty acids in triacylglycerol samples of rabbit mammary
_
and non-esterified tissue taken during
fatty acid fractions involution
6:0 8:O to:0 12:o 14:O 16:O 16:l 18:O 1x:1 18:2 18:3
(ii 0.7 * 0.1 32.5 23.2 4.0 2.0 12.4 2.9 2.1 9.3 10.5 1.3
Molar proportions in parentheses. * P < 0.05. **P < 0.01.
2 5.5 i 3.8 & 0.9 ;t 0.3 + 3.3 * 1.1 5 0.4 & 2.6 rt 2.0 -+ 0.3
1.0 i: 0.1 31.6 20.4 3.5 2.0 12.7 2.4 2.7 11.0 10.3 3.3
of individual
i: 3.6 ri: 2.3 If: 0.4 + 0.3 & 1.9 tr: 0.9 ir: 0.5 I_ 1.7 & 1.3 5 0.5
i: rt: & t. & + + + & +
(G,
t49
0.8 + 0.1 26.4 19.9 3.4 2.2 14.6 2.1 2.4 10.6 16.4 4.2
5.3 2.9 0.4 0.4 2.8 0.7 0.1 1.3 4.5 1.1
fatty acids significantiy
0.7 2 0.1 19.9 12.7 1.7 3.1 19.9 3.5 3.4 15.8 16.4 3.4
from biopsy
Non-esterified fatty acid fraction (mol %I
Triacylglycerol fraction (mot %) Day Fatty acid
isolated
f + f + 5 + + + + +
different
5.1 3.1* 0.5 0.5* 2.9** 1.8 0.2 2.2* 2.2* 0.5
0.06 * 0.02** 2.9 4.4 1.6 3.2 23.6 6.5 5.8 25.8 24.3 1.6
& 1.3** + 0.6** c 1.0 i 0.3* f i.i+ + 2.1 +_ 1.5 _t l.O** + 1.5** j, 0.6
from Day 0. Mean
:P,
1.8 3.1 5.0 7.8 40.2 3.9 23.9 9.1 4.9 2.0
-
i: 0.5 + 1.4 + 1.6 +_ 1.0 + 0.7 * 0.7 t 1.9 + 1.4 + 1.5 + 0.8
+ SE, number
4.3 5.0 4.0 7.1 40.8 5.6 15.2 12.0 4.8 0.5
of animals
It: + + 4 f + f + f f
2.6 2.2 0.5 0.3 2.4 2.2 3.1 1.0 0.4 0.2
sampled
Involution
of rabbit
that of the TAG fraction did. It can be seen that the medium chain fatty acids C8:O and C1O:O decreased as a molar percentage of the total fatty acids in the TAG fraction reflecting the reduction in the activity of the enzyme fatty acid synthetase during this time. There was no indication that the medium chain fatty acids enter the NEFA pool in the gland which suggests that TAG fatty acids may be removed from the gland into the lymph by macrophages as suggested by Lee et al. (1969) rather than into the blood following lipolysis within the gland. The normal behaviour of the female rabbit results in feeding the young only once every 24 hr. Therefore, in our study it would be expected that any control mechanism which affects the activity in the gland would be initiated after this time. The period of rapid change reported in the present data is apparent between 48 and 72 hr after removal of the young at a time when milk accumulation is at a maximum (Ota & Peaker, 1979). The nature of the stimuli which regulate the metabolic activity within the gland during involution are not well understood. The rapid fall in blood flow to the gland following removal of young as demonstrated in the rat (Hanwell & Linzell, 1973) will affect the hormonal status of the tissue and also the supply of metabolites to the gland. Earlier studies in the rabbit (Mena at al., 1974) suggested that blood prolactin levels may be a regulatory factor although studies with thelectomised rats (Moore 8z Forsyth, 1980) where blood hormone levels were unchanged suggest that it is the local concentration of hormone at the tissue level that may be more important. In our studies there was no reduction in plasma prolactin levels measured in arterial and mammary venous blood (Jones & Parker, 1982) during the period studied although this will not reflect the situation within the tissue. A feedback effect by metabolites produced within the gland on both activity within the mammary gland and capillary blood flow has been suggested from studies in the goat (Peaker, 1980) and has also been implicated in the control of amino acid uptake by the rat mammary gland during initiated involution (Vina et al., 1981). The interaction between milk accumulation and other factors such as blood flow and hormonal changes has yet to be elucidated.
ied whereas
AcknoMlrdgrmcnt-This from the Agricultural
work was supported Research Council.
by a grant
REFERENCES
ASH R. & BAIRD G. D. (1973) Activation of volatile fatty acids in bovine liver and rumen epithelium. Biochem. J. 136, 31 I-319. BAUMAN D. E., MELLENRERCER R. W. & INGLE D. (1974) Metabolic adaptation in fatty acid and lactose biosynthesis by sheep mammary tissue during cessation of lactation. J. Dairy Sci. 51, 719-723. BERGMEYERH. U. & BRENT E. (1974) In Methods ofEnzymatic Analysis (BERGMEYERH. U., Ed.) pp. 5755579. Academic Press. New York.
mammary
gland
569
CRABTREE B., TAYLOR D. J., COOMBS J. E.. SMITH R. A., TEMPLER S. P. & SMITH G. H. (1981) The activities and intracellular distributions of enzymes of carbohydrate, lipid and ketone-body metabolism in lactating mammary glands from ruminants and non-ruminants. Biothem. J. 196, 747-756. GUL B. & DILS R. R. (1969) Enzymic changes in rabbit and rat mammary gland during the lactation cycle. Biochem. J. 112, 293-301. HAMOSH M., CLARY T. R., CHERNICK S. S. & Scow R. 0. (1970) Lipoprotein lipase activity of adipose and mammary tissue and plasma triglyceride in pregnant and lactating rats. Biochim. hiphys. Acta 210, 473-482. HANWELL A. & LINZELL J. L. (1973) The effects of engorgement with milk and of suckling on mammary blood flow in the rat. J. Phpsiol., Lond. 233, 11 t-125. HUANG K. P. (1970) A sensitive assay method for acetyl CoA synthetase. Analyt. Eiochrm. 37, 98-104. JONES E. A. (1967) Changes in the enzyme pattern of the mammary gland of the lactating rat after hypophysectomy and weaning. Biochem. J. 103, 42&427. JONES E. A. (1968) The relationship between milk accumulation and enzyme activities in the involuting rat mammary gland. Biochim. hiophys. Acta 177, 158-160. JONES C. S. & PARKER D. S. (1978) Uptake of substrates for milk fat synthesis by lactating-rabbit mammary gland. Biochem. J. 174, 291-296. JONES C. S. & PARKER D. S. (1981) The metabolism of glucose, acetate and palmitate in the lactating rabbit. Comp. Biochem. Physiol. 69B, 837-842. JONES C. S. & PARKER D. S. (1982) Submitted for publication. LASCELLES A. K. & LEE C. S. (1978) Involution of the mammary gland. In Lactation Vohtme IV (LARSON B. L.. ED.) pp. 115-177. Academic Press, New York. LEE C. S., MCDOWELL G. H. & LASCELLESA. K. (1969) The importance of macrophages in the removal of fat from the involuting mammary gland. Res. Vet. Sci. 10. 3438. MARTIN D. B., HORNING M. G. & VAGELOS P. R. (1961) Fatty acid synthesis in adipose tissue. J. hiol. Chem. 236, 663-668. MCBRIDE 0. W. & KORN E. D. (1963) The lipoprotein lipase of mammary gland and the correlation of its activity to lactation. J. Lipid Res. 4, 17-20. MENA F., REYES G., AGUAYO D. & GROSVENOR C. E. (1974) Effect of acute reduction in milk removal from some glands upon the ability of a single rabbit mammary gland to secrete milk J. Endocrin. 62, 431-438. MOORE B. P. & FORSYTH I. A. (1980) Influence of local vascularity on hormone receptors in mammary gland. Nuture 284, 77-78. MWRE J. H. & CHRISTIE W. W. (1981) Lipid metabolism in the mammary gland of ruminant animals. In Lipid Metabolism in Ruminant Animals (CHRISTIE W. W., Ed.) pp. 227-278. Pergamon Press. Oxford. OTA K. & PEAKER M. (1979) Lactation in the rabbit: Mammary blood flow and cardiac output. Q. J. exp. Physiol. 64, 225-238. PEAKER M. (1980) The effect of raised intramammary pressure on mammary function in the goat in relation to the cessation of lactation. J. Physiol. 301, 415-428. SCHMIDT E. (1974) In Methods of Enzymatic Analysis (BERGMEYER H. U., Ed.) pp. 65&656. Academic Press, New York. SRERE P. A., BRAZIL H. & GONEN L. (1963) The citrate condensing enzyme of pigeon breast muscle and moth flight muscle. Acta them. scud. 17, S129-S134. VINA J. R.. PUERTES 1. R. & VINA J. (1981) Effect of premature weaning on amino acid uptake by the mammary gland of lactating rats. Biochem. J. 200, 7055708.
Biochrnl. in Great
Vol.
IS, No. 4, pp. 565-569.
Britain.
All rights
1983
0020.7
reserved
CopyrIght
0
I IXi83:040565-05$03.00/O 1983 Pergamon
Press
Ltd
THE EFFECT OF INITIATED INVOLUTION ON ENZYME ACTIVITY AND SUBSTRATE UPTAKE BY THE MAMMARY GLAND OF THE LACTATING RABBIT COLIN S. JONES* and DAVID S. PARKERt Department of Physiology and Biochemistry, University of Reading, Whiteknights, Reading RG6 2AJ, U.K. 13 July 1982)
(Received
Arteriovenous difference studies across the lactating rabbit mammary gland for glucose, acetate, triacylglycerol and non-esterified fatty acids during initiated involution are reported. 2. A significant reduction in substrate utilisation is paralleled by a decrease in the activities of fatty acid synthetase, acetyl CoA synthetase, citrate synthase and glutamate dehydrogenase in biopsy samples
Abstract--l.
taken from the gland. 3. Results from the analysis
of linid fractions
within
the gland
during
this period
are discussed
in
relation to lipid resorption.
INTRODUCTION
aseptically from a separate group of does. A small incision was made in the skin covering one of the abdominal glands and tissue excised using diathermy. The tissue was homogenised in 0.25 M sucrose and fractionated into sub-cellular components by the method described by Ash & Baird (1973). Fractions were stored at -20” prior to assay for specific enzymes. All assay procedures were checked for linearity with time and protein concentration; the individual assay systems used were: glutamate dehydrogenase (Schmidt, 1974); fatty acid synthetase (Martin et al., 1961); citrate synthase (Srere et al., 1963); acetyl CoA synthetase (Huang, 1970) and lactate dehydrogenase (Bergmeyer & Brent, 1974). Tissue lipids were separated into individual components by t.1.c. and the TAG and NEFA fractions prepared for g.1.c. (Jones & Parker, 1978).
Mammary gland arteriovenous difference studies and blood flow measurements in the lactating rabbit have demonstrated the importance of various metabolites in the metabolism of the gland and as precursors for milk synthesis (Jones & Parker, 1978; Jones & Parker, 1981). During initiated involution of the mammary gland brought about by the premature removal of the young, there has been shown to be a rapid reduction in metabolic activity within the gland of the rat (Jones, 1967, 1968) and the ewe (Bauman et al., 1974). This is followed by regression of the mammary tissue and resorption of the milk constituents (see review by Lascelles & Lee, 1978). The following experiments were designed to investigate the response of the lactating rabbit mammary gland to initiated involution with reference to metabolite uptake by the gland and also tissue enzyme studies and lipid content measured in biopsy samples.
RESULTS AND DlSCUSSlON The activities of four of the enzymes studied show a significant fall immediately after the removal of the young. This is reflected in both the mitochondrial glutamate dehydrogenase and citrate synthase activities and the cytosolic enzymes fatty acid synthetase and acetyl CoA synthetase (Fig. 1). The activity of lactate
METHODS Lactating New Zealand White rabbits (Buxted Rabbits, Buxted, Sussex) were obtained at around day 14 of lactation and housed under conditions of 16 hr day length.
dehydrogenase (33.0 + 2.6 (21) pmol substrate converted/min per g wet wt tissue) did not change during the period studied. These results indicate a rapid response within the tissue to the cessation of milk removal and are in agreement with data for other enzymes in the rat (Jones, 1967, 1968), the sheep (Bauman et ul., 1974) and one other study in the rabbit (GUI & Dils, 1969). In addition to changes in the activity it is also of interest that the intracellular distribution of both the mitochondrial enzymes changed during the course of the period studied, with increased activity being detected in the cytosolic fraction as involution progressed. This may indicate increased leakage of enzymes from mitochondria as cell lysis progresses during involution. Histological examination of tissue taken from rabbits during this study show the presence of macrophages and cytosegresomes as has been reported in sheep and rat involuting tissue (Lascelles & Lee, 1978).
Food (Diet 18, Dixon & Sons, Ware, Herts) and water were available ad lihitum. The day that the young were removed from the doe was designated Day 0 of involution. The carotid artery and both lateral thoracic veins were cannulated as previously described (Jones & Parker, 1978). Blood sampling commenced within 48 hr and samples were obtained once a day for as long as the cannulae remained open. Blood samples were analysed for glucose, acetate, triacylglycerol (TAG) and non-esterified fatty acids (NEFA) as previously described (Jones & Parker, 1978). Biopsy samples of mammary tissue (500 mg) were obtained
* Present address: Department of Zoology, University of Durham, Durham. t Present address: Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, Newcastle upon Tyne. 565
COLIN S. JONES and DAVIU
S. PARKER
Acetyl CoA synthetose 6.0/-
--
2
4
6 Days after
8
Fatty
of the
synthetase
4
2
IO
removal
acid
6
IO
young
Fig. 1. Activity of rabbit mammary gland enzymes assayed in biopsy samples taken during invoiution. Activity expressed as pmol substrate converted/min per g wet wt of tissue, mean f SE. In all cases enzyme activities determined from Day 3 to Day 10 were significantly different (P c: 0.1) from that determined on Day 0. Number of biopsy samples; Day 0 = 4, Day 2 = 4, Day 3 = 4. Day 6 = 3. Day 8 = 3, Day 10 = 3.
Arteriovenous difference studies for the mammary gland underline the changes in metabolic activity occurring after removal of the pups. The results for glucose, acetate, TAG and NEFA are shown in Fig. 2. Uptake of both glucose and acetate falls rapidly within 48 hr of the removal of the young and from then until 10 days of involution remains almost zero. Both glucose and acetate are utilised extensively in the lactating rabbit mammary gland for lipid synthesis and oxidative pathways (Crabtree et al., 1981). Studies using labelled substrate indicate that these two metabolites account for SO”/, of total CO2 output by the lactating gland (Jones & Parker, 1981) and the
reduction in uptake during involution reflects the fall in metabolic activity within the gland at this time. The arteriovenous difference values for TAG also show a reduction in uptake by the gland in the first 2 days of involution although the data for some of the later samples shows an apparent reversal of this trend. These mean values have large standard errors associated with them due to some individual rabbits having very high plasma TAG values. The data for NEFA are, however, much clearer and show a very interesting pattern. The uptake of NEFA by the lactating gland noted in a previous study (Jones & Parker, 1978) changes to a net output of NEFA by the gland
Involution
of rabbit
mammary
gland
561
I2H E
IO08
-
i 8
06-
1 3
04
-
0.2
-
Uptake Output
L
t
0
2 Days
4 after
6 removal
8 of
the
IO young
Fig. 2. Arteriovenous difference values for glucose, acetate, TAG and NEFA across the rabbit mammary gland during involution. Mean LSE, dotted line indicates no net uptake or output by the mammary gland. Number of individual samples analysed were as follows; Day 0 = 10, Days 1, 2 = 2, Day 3 = 7, Day 4 = 8, Days 5, 6 = 6, Days 7, 8 = 5, Day 9 = 4, Day 10 = 3.
48 hr after removal of the young. This reaches a maximum at about 72 hr and then remains constant for the remainder of the period studied. The results of analysis of the individual fatty acids within the NEFA fraction (Fig. 3) show that this increase is primarily in the C16:0, C18:O and C18: 1 fatty acids and that the medium chain length fatty acids C8:O and ClO:O characteristic of rabbit milk are not released into the venous blood. NEFA present in mammary venous blood during lactation are derived from both the arterial supply to the gland and also from fatty acids released from plasma TAG following the action of lipoprotein lipase on the endothelial cell surface
(Moore & Christie, 1981). During involution, however, the activity of this enzyme has been shown to decline rapidly in both guinea-pig (McBride & Korn, 1963) and rat (Hamosh et al., 1970) mammary glands following removal of the young. The positive arteriovenous differences reported in the present study may, therefore, reflect loss of fatty acids from the cells of the involuting rabbit mammary gland. Tissue lipid extracted from biopsy samples during the same period were separated into NEFA and TAG fractions and the individual fatty acids from each fraction are shown in Table 1. The composition of the NEFA fraction did not alter during the 10 days stud-
COLIN S. JONESand DAVID S. PARKER
568
RUptake
Days
after
removal
of
the
young
Fig. 3. Arteriovenous difference values (PM) for individual plasma NEFA across the rabbit mammary gland during involution. Mean + SE, dotted line indicates no net uptake or output by the mammary gland. Number of individual samples analysed were as follows; Day 0 = 10, Days 1, 2 -i 2, Day 3 = 7, Day 4 = 8. Days 5, 6 = 5, Day 9 = 4, Day 10 = 3.
Table
1. Molar
proportions
of fatty acids in triacylglycerol samples of rabbit mammary
_
and non-esterified tissue taken during
fatty acid fractions involution
6:0 8:O to:0 12:o 14:O 16:O 16:l 18:O 1x:1 18:2 18:3
(ii 0.7 * 0.1 32.5 23.2 4.0 2.0 12.4 2.9 2.1 9.3 10.5 1.3
Molar proportions in parentheses. * P < 0.05. **P < 0.01.
2 5.5 i 3.8 & 0.9 ;t 0.3 + 3.3 * 1.1 5 0.4 & 2.6 rt 2.0 -+ 0.3
1.0 i: 0.1 31.6 20.4 3.5 2.0 12.7 2.4 2.7 11.0 10.3 3.3
of individual
i: 3.6 ri: 2.3 If: 0.4 + 0.3 & 1.9 tr: 0.9 ir: 0.5 I_ 1.7 & 1.3 5 0.5
i: rt: & t. & + + + & +
(G,
t49
0.8 + 0.1 26.4 19.9 3.4 2.2 14.6 2.1 2.4 10.6 16.4 4.2
5.3 2.9 0.4 0.4 2.8 0.7 0.1 1.3 4.5 1.1
fatty acids significantiy
0.7 2 0.1 19.9 12.7 1.7 3.1 19.9 3.5 3.4 15.8 16.4 3.4
from biopsy
Non-esterified fatty acid fraction (mol %I
Triacylglycerol fraction (mot %) Day Fatty acid
isolated
f + f + 5 + + + + +
different
5.1 3.1* 0.5 0.5* 2.9** 1.8 0.2 2.2* 2.2* 0.5
0.06 * 0.02** 2.9 4.4 1.6 3.2 23.6 6.5 5.8 25.8 24.3 1.6
& 1.3** + 0.6** c 1.0 i 0.3* f i.i+ + 2.1 +_ 1.5 _t l.O** + 1.5** j, 0.6
from Day 0. Mean
:P,
1.8 3.1 5.0 7.8 40.2 3.9 23.9 9.1 4.9 2.0
-
i: 0.5 + 1.4 + 1.6 +_ 1.0 + 0.7 * 0.7 t 1.9 + 1.4 + 1.5 + 0.8
+ SE, number
4.3 5.0 4.0 7.1 40.8 5.6 15.2 12.0 4.8 0.5
of animals
It: + + 4 f + f + f f
2.6 2.2 0.5 0.3 2.4 2.2 3.1 1.0 0.4 0.2
sampled
Involution
of rabbit
that of the TAG fraction did. It can be seen that the medium chain fatty acids C8:O and C1O:O decreased as a molar percentage of the total fatty acids in the TAG fraction reflecting the reduction in the activity of the enzyme fatty acid synthetase during this time. There was no indication that the medium chain fatty acids enter the NEFA pool in the gland which suggests that TAG fatty acids may be removed from the gland into the lymph by macrophages as suggested by Lee et al. (1969) rather than into the blood following lipolysis within the gland. The normal behaviour of the female rabbit results in feeding the young only once every 24 hr. Therefore, in our study it would be expected that any control mechanism which affects the activity in the gland would be initiated after this time. The period of rapid change reported in the present data is apparent between 48 and 72 hr after removal of the young at a time when milk accumulation is at a maximum (Ota & Peaker, 1979). The nature of the stimuli which regulate the metabolic activity within the gland during involution are not well understood. The rapid fall in blood flow to the gland following removal of young as demonstrated in the rat (Hanwell & Linzell, 1973) will affect the hormonal status of the tissue and also the supply of metabolites to the gland. Earlier studies in the rabbit (Mena at al., 1974) suggested that blood prolactin levels may be a regulatory factor although studies with thelectomised rats (Moore 8z Forsyth, 1980) where blood hormone levels were unchanged suggest that it is the local concentration of hormone at the tissue level that may be more important. In our studies there was no reduction in plasma prolactin levels measured in arterial and mammary venous blood (Jones & Parker, 1982) during the period studied although this will not reflect the situation within the tissue. A feedback effect by metabolites produced within the gland on both activity within the mammary gland and capillary blood flow has been suggested from studies in the goat (Peaker, 1980) and has also been implicated in the control of amino acid uptake by the rat mammary gland during initiated involution (Vina et al., 1981). The interaction between milk accumulation and other factors such as blood flow and hormonal changes has yet to be elucidated.
ied whereas
AcknoMlrdgrmcnt-This from the Agricultural
work was supported Research Council.
by a grant
REFERENCES
ASH R. & BAIRD G. D. (1973) Activation of volatile fatty acids in bovine liver and rumen epithelium. Biochem. J. 136, 31 I-319. BAUMAN D. E., MELLENRERCER R. W. & INGLE D. (1974) Metabolic adaptation in fatty acid and lactose biosynthesis by sheep mammary tissue during cessation of lactation. J. Dairy Sci. 51, 719-723. BERGMEYERH. U. & BRENT E. (1974) In Methods ofEnzymatic Analysis (BERGMEYERH. U., Ed.) pp. 5755579. Academic Press. New York.
mammary
gland
569
CRABTREE B., TAYLOR D. J., COOMBS J. E.. SMITH R. A., TEMPLER S. P. & SMITH G. H. (1981) The activities and intracellular distributions of enzymes of carbohydrate, lipid and ketone-body metabolism in lactating mammary glands from ruminants and non-ruminants. Biothem. J. 196, 747-756. GUL B. & DILS R. R. (1969) Enzymic changes in rabbit and rat mammary gland during the lactation cycle. Biochem. J. 112, 293-301. HAMOSH M., CLARY T. R., CHERNICK S. S. & Scow R. 0. (1970) Lipoprotein lipase activity of adipose and mammary tissue and plasma triglyceride in pregnant and lactating rats. Biochim. hiphys. Acta 210, 473-482. HANWELL A. & LINZELL J. L. (1973) The effects of engorgement with milk and of suckling on mammary blood flow in the rat. J. Phpsiol., Lond. 233, 11 t-125. HUANG K. P. (1970) A sensitive assay method for acetyl CoA synthetase. Analyt. Eiochrm. 37, 98-104. JONES E. A. (1967) Changes in the enzyme pattern of the mammary gland of the lactating rat after hypophysectomy and weaning. Biochem. J. 103, 42&427. JONES E. A. (1968) The relationship between milk accumulation and enzyme activities in the involuting rat mammary gland. Biochim. hiophys. Acta 177, 158-160. JONES C. S. & PARKER D. S. (1978) Uptake of substrates for milk fat synthesis by lactating-rabbit mammary gland. Biochem. J. 174, 291-296. JONES C. S. & PARKER D. S. (1981) The metabolism of glucose, acetate and palmitate in the lactating rabbit. Comp. Biochem. Physiol. 69B, 837-842. JONES C. S. & PARKER D. S. (1982) Submitted for publication. LASCELLES A. K. & LEE C. S. (1978) Involution of the mammary gland. In Lactation Vohtme IV (LARSON B. L.. ED.) pp. 115-177. Academic Press, New York. LEE C. S., MCDOWELL G. H. & LASCELLESA. K. (1969) The importance of macrophages in the removal of fat from the involuting mammary gland. Res. Vet. Sci. 10. 3438. MARTIN D. B., HORNING M. G. & VAGELOS P. R. (1961) Fatty acid synthesis in adipose tissue. J. hiol. Chem. 236, 663-668. MCBRIDE 0. W. & KORN E. D. (1963) The lipoprotein lipase of mammary gland and the correlation of its activity to lactation. J. Lipid Res. 4, 17-20. MENA F., REYES G., AGUAYO D. & GROSVENOR C. E. (1974) Effect of acute reduction in milk removal from some glands upon the ability of a single rabbit mammary gland to secrete milk J. Endocrin. 62, 431-438. MOORE B. P. & FORSYTH I. A. (1980) Influence of local vascularity on hormone receptors in mammary gland. Nuture 284, 77-78. MWRE J. H. & CHRISTIE W. W. (1981) Lipid metabolism in the mammary gland of ruminant animals. In Lipid Metabolism in Ruminant Animals (CHRISTIE W. W., Ed.) pp. 227-278. Pergamon Press. Oxford. OTA K. & PEAKER M. (1979) Lactation in the rabbit: Mammary blood flow and cardiac output. Q. J. exp. Physiol. 64, 225-238. PEAKER M. (1980) The effect of raised intramammary pressure on mammary function in the goat in relation to the cessation of lactation. J. Physiol. 301, 415-428. SCHMIDT E. (1974) In Methods of Enzymatic Analysis (BERGMEYER H. U., Ed.) pp. 65&656. Academic Press, New York. SRERE P. A., BRAZIL H. & GONEN L. (1963) The citrate condensing enzyme of pigeon breast muscle and moth flight muscle. Acta them. scud. 17, S129-S134. VINA J. R.. PUERTES 1. R. & VINA J. (1981) Effect of premature weaning on amino acid uptake by the mammary gland of lactating rats. Biochem. J. 200, 7055708.