Wahle, K. W. J., Morris, J. J. & Garton, G. A. (1974) Biochem. SOC. Truns. 2, 598-599. Hormonal Control of Lipogenesis: the Induction of Milk-Fat Synthesis in.
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Vernon, R. G. (1974) Biochern. SOC.Trans. 2,142-143 Viviani, R. (1970) Advan. Lipid Res. 8,267-346 Wadsworth, J. C. (1968) J. Dairy Sci.51, 876-881 Wahle, K. W. J. & Davies, N. T. (1974) Eiochem. SOC.Trans. 2, 1283-1285 Wahle, K. W. J., Morris, J. J. & Garton, G. A. (1974) Biochem. SOC.Truns. 2, 598-599
Hormonal Control of Lipogenesis: the Induction of Milk-Fat Synthesis in Tissue Explants of Mammary Gland R. DILS, B. SPEAKE, R. J. MAYER and E. LYNCH Department of Biochemistry, University of Nottingham, Nottingharn NG7 2RD, U.K., and C . R. STRONG Department of Biochemistry, University of St. Andrews, Fife KY16 9AL, U.K., and I. A. FORSYTH National Institute for Research in Dairying, Shinfield, Reading RG2 9AT, U.K.
Most mammalian tissues contain predominantly long-chain (>CI4) fatty acids. By contrast, the milk fat from many species contains considerable proportions of short(C4: and Cs:o ) and medium-chain (C, :o-Clz :o ) fatty acids esterified as triglyceride. There is evidence that in a number of species the medium-chain fatty acids are synthesized within the mammary gland. This raises a number of questions. For example, when are these fatty acids first synthesized by the mammary gland in the lactation cycle, and what hormones induce the onset of their synthesis? We have found that rabbit mammary gland is an excellent tissue to work with in attempting to answer these questions. The rabbit is pregnant for 30-31 days. In early pregnancy (up to day 18) the mammary gland synthesizes predominantly long-chain (C14:o-Cls: 1) fatty acids. Between day 18 and day 22 of pregnancy there is a striking increase (up to 12-fold) in the rate of fatty acid synthesis/g wet wt. of tissue. This increase is due almost entirely to a sudden onset in the synthesis of C , : and Clo: acids, which are characteristic of rabbit milk (Smith et al., 1968). The fatty acids synthesized by the gland throughout the remaining period of pregnancy are almost exclusively these medium-chain fatty acids. At or near parturition there is a second lipogenic stimulus, though the gland continues to synthesize only C, : and Clo: acids in lactation (Carey & Dils, 1972; Strong & Dils, 1972; Mellenberger & Bauman, 1974). This period between day 18 and 22 of pregnancy, when milk-fat synthesis first occurs in the rabbit, is especially interesting. Bousquet et al. (1969) have found that there are marked changes in the ultrastructure of the epithelial cells of the gland during this period. These changes indicate a considerable development of the cellular structures involved in the synthesis and secretion of milk. For example, by day 22 of pregnancy the cytoplasm contains dilated vesicles of endoplasmic reticulum and abundant mitochondria. Lactose becomes detectable in the gland (Denamur, 1963) and the lumen contains protein granules, which are probably casein. There is therefore considerable evidence for the onset of lactogenesis during this period. Hormonal control of lipogenesis in rabbit mammary gland The hormonal initiation of lactogenesis in the mammary glands of pregnant mice has been extensively investigated by using the technique of organ culture [see Forsyth (1971) for review]. This technique enables small pieces (explants) of the mammary tissue, usually l-2mm3, to be maintained in vitro in a controlled environment for a number of days so that the architecture and function of the tissue is maintained as far as possible. It is then possible to measure which hormones, either singly or in combination, can initiate and maintain lactogenesis in mammary explants from pregnant animals. Most of the work done using this technique has been concerned with th6 hormonal induction of the synthesis of milk proteins and lactose synthetase in mid-pregnant mouse mammary gland. By contrast, theeffects ofhormones on thelipogenicactivity ofmammary explants
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has received much less attention. Moretti & Abraham (1966) found that insulin stimulates fatty acid synthesis from glucose by mammary-gland explants from mid-pregnant mice. However, the rate of synthesis is better maintained over 2 days in culture with insulin, corticosterone and prolactin (Mayne & Barry, 1970), and lipid droplets can be observed histologically in mammary explants with this hormone combination. These results led us to examine (Forsyth et al., 1972) the effects of these hormones on the rate and pattern of fatty acids synthesized by mammary explants from rabbits in early pregnancy. As expected, mammary explants from rabbits at day 16 of pregnancy synthesized long-chain fatty acids from W-labelled acetate plus glucose. After culture for 2 to 4 days with insulin, corticosterone and prolactin the overall rate of fatty acid synthesis increased 3 0 - f o l d and gave rates of lipogenesis similar to those in early lactation. However, the proportion of medium-chain fatty acids synthesized was sometimes higher when corticosterone was omitted from the culture. Although insulin increased the overall rate of fatty acid synthesis and improved the viability of the tissue, it did not affect the pattern of fatty acids synthesized. The most striking results were obtained when explants were cultured with prolactin alone for 2 to 4 days. This caused a 10-15-fold increase in the rate of lipogenesis, which is the same as that occurring between day 18 and day 22 of pregnancy in rabbit mammary gland. Further, the pattern of fatty acids synthesized changed so that Cs: and Clo: were the predominant fatty acids synthesized. Ultrastructural studies also indicated that dilated rough endoplasmic reticulum had increased and that casein-like protein granules appeared in the epithelial cells and alveolar lumina. Essentially similar results have been obtained by using mammary explants from rabbits, which had been pseudopregnant for 11 days. Again, culturing with prolactin alone induced the synthesis of medium-chain fatty acids (Strong et al., 1972). In summary, with rabbit mammary explants only prolactin is required to induce the synthesis of medium-chain fatty acids characteristic of rabbit milk. Whether casein and lactose synthesis respond in a similar manner in this system awaits further investigation. These results contrast considerably with earlier reports of the hormonal induction of casein and lactose synthesis by explants from mid-pregnant mouse mammary gland [see Topper (1970) for review]. Culturing with insulin stimulated the epithelial cells to synthesize DNA and to divide. The daughter cells are then sensitive to cortisol, which stimulated the production of rough endoplasmicreticulum. Only then could the epithelial cells respond to the synergistic actions of insulin and prolactin by synthesizing casein and lactose. Though these interpretations have recently been somewhat modified (Oka & Topper, 1972; Owens et af., 1972; Mukherjee et af., 1973) it appears that the induction of milk-fat synthesis in mid-pregnant rabbit mammary gland is under much simpler hormonal control. This difference has been emphasized in that mammary explants from 16-day pregnant rabbits and from 11-day pseudopregnant rabbits can synthesize milk fat in response to prolactin even in the presence of hydroxyurea (which inhibits thymidine incorporation in our system). It therefore appears that DNA synthesis is not a prerequisite for milk-fat synthesis in the rabbit mammary gland. Turnover of fatty acid synthetase in fabbit mammary gland As described above, there is an increase in the synthetic capacity of rabbit mammary gland between days 18 and 22 of pregnancy. The increase in the rate of lipogenesis can be mimicked by culturing mammary explants with hormones. This appeared to be a good system with which to study the effects of hormones on the turnover of enzymes involved in the increased lipogenesis. In addition the system can be manipulated to mimic the process of involution in the mammary gland by subsequently removing the explants from the hormonal stimulus for lactogenesis. Our efforts so far have been concentrated on studying the turnover of fatty acid synthetase in this system. When mammary explants from 16-day pregnant rabbits were cultured with insulin, corticosterone and prolactin, there was a 1 0 - f o l d increase in the specific activity of fatty acid synthetase after 40h in culture. Enzyme activity was measured by the malonylCoA-dependent oxidation of NADPH. When maximum stimulation of overall lipo1974
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Culture time (h) Fig. 1. Efects of hormones on the amount andactivity of fatty acid synthetase in mammary explants during culture Mammary explants from 16-daypregnant rabbits werecultured at 37°C in an atmosphere of COz+Oz (5:95) in Medium 199 (Strong et a/., 1972) containing insulin (5pg/ml), corticosterone (1 pg-ml/) and prolactin (1 pg/ml). After culturing for the times shown, 40 explants were removed and homogenized in 0.25 ex potassium phosphate buffer, pH7.0, containing 1mM-dithiothreitol. The homogenate was centrifuged at 106g,,.-min. The supernatant (which contained approx. 1-2mg of protein/ml) was assayed for fatty acid synthetase activity by the method of Carey & Dils (1970). Immunological titration of fatty acid synthetase was carried out by adding increasing amounts of anti-(fatty acid synthetase) y-globulin ot 0.1 ml portions of the supernatant. The mixture was incubated at 37°C for lOmin and then allowed to remain for 24h at 4°C. The samples were then assayed for fatty acid synthetase activity as described above. 0 , Fatty acid synthetase activity of explants cultured with hormones; A, fatty acid synthetase activity of explants after removal of hormones; 0 , immunologically detectable fatty acid synthetase in explants cultured with hormones; A, immunologically detectable fatty acid synthetase in explants after removal of hormones.
genesis had been attained, the explants were transferred to hormone-free culture medium. This resulted in a dramatic decline in the specific activity of the fatty acid synthetase. The specific activity fell within 30h to that observed in explants that had not been cultured with hormones. We have raised antibodies in sheep and fowl to purified rabbit mammary-gland fatty acid synthetase. Immunological titrations with these anti(fatty acid synthetase) antibodies showed that the changes in the specific activity of fatty acid synthetase can be explained by changes in the amount of the enzyme in the explants rather than by changes in the activity of a constant amount of the enzyme. In the presence of the three hormones, the rate of lipogenesis in the explants (measured bytheincorporationof [l-14C]acetate into fatty acids) increased to a maximum after 20h in culture. By contrast, the maximum increase in the amount of fatty acid synthetase occurred after 40h in culture. Removal of the hormones caused a precipitous decrease within 4h in the overall rate of lipogenesis to half of the maximum value. The amount of fatty acid synthetase present in the explants decreased to half the maximum amount in approx. 15h (Fig. 1). It is interesting that the increase in the rate of synthesis of fatty acid synthetase in the presence of insulin, corticosterone and prolactin closely paralleled the increase in the rate of synthesis of total cytosolic protein (these rates were measured
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by immunoprecipitation of fatty acid synthetase and by trichloroacetic acid precipitation of cytosolic protein after the incubation of explants with [U-14C]leucine). These preliminary results indicate the usefulness of this system as a model with which to study the hormonal control of the amounts of enzymes in developing tissues. Hormonal control of lipogenesis in goat mammary gland
The technique of organ culture does not appear to have been used to study lipogenesis in ruminant mammary gland. We have therefore begun to investigate the hormonal control of lipogenesis in explants from goat mammary gland. The approach has been similar to that described for rabbit mammary gland. Mammary tissue has been obtained by biopsy techniques from goats, which have been pregnant for 61,74,81,90,111 and 134 days and from goats during lactation. The maximum increase in the overall rate of lipogenesis was obtained when explants were cultured for 1 to 2 days with insulin (5pg/ml), cortisol (hydrocortisone-21 acetate) (1 ,ug/ml) and prolactin (1 ,ug/ml). The lipogenic response of the tissue to this hormone combination decreased throughout pregnancy. With 61-day pregnant goats there was a 7.5-fold increase in the rate of lipogenesis. With 134-day pregnant and with lactating animals there was little or no response to the hormones (E. Lynch, E. F. Annison & R. Dils, unpublished work). It will be of interest to know whether these hormones affect the proportions of fatty acids synthesized so that they resemble the proportions found in goat milk. Bousquet, M., Flechon, J. E. &Denamur, R. (1969)Z.ZelZjiorsch. Mikrosk. Anat. 96,418-436 Carey, E. M. & Dils, R. (1970) Biochim. Biophys. Acta 210,371-387 Carey, E. M. & Dils, R. (1972) Biochem. J. 126,1005-1007 Denamur, R. (1963) C. R. Acad. Sci. Ser. D 256,47484752 Forsyth, I. A. (1971)J. Dairy Res. 3 , 4 1 9 4 5 Forsyth, I. A., Strong, C. R. & Dils, R. (1972) Biochern. J. 129,929-935 Mayne, R. & Barry, J. M. (1970) J. Endocrinol. 46,61-70 Mellenberger, R. W. & Bauman, D. E. (1974) Biochem. J . 138,373-379 Moretti, R. L. & Abraham, S. (1966) Biochim. Biophys. Acta 124,280-288 Mukherjee, A. S., Washburn, L. L. & Banerjee, M. R. (1973) Nature (London)256, 159-160 Oka, T. &Topper, Y. J. (1972) Proc. Nat. Acad. Sci. U.S.69, 1693-1696 Owens, I. S., Vonderhaar, B. K. & Topper, Y . J (1973) J. Biol. Chem. 248,472-477 Smith, S., Watts, R. & Dils, R. (1968) J . Lipid Res. 9, 52-57 Strong, C. R. & Dils, R. (1972) Biochem. J. 128, 1303-1309 Strong, C. R., Forsyth, I. & Dils, R. (1972) Biochem. J. 128,509-519 Topper, Y. J. (1970) Recent. Progr. Horm. Res. 26,287-308
Acute Hormonal Regulation of Fatty Acid Synthesis in Rat Epididymal Fat-Pads RICHARD M. DENTON Department of Biochemistry, University of Bristol, Medical School, University Walk, Bristol BS8 1 T D , U.K. Introduction
Brief exposure to insulin results in a striking increase in the rate of fatty acid synthesis from glucose in rat epididymal fat-pads. The increase is often in excess of fivefold, whereas the associated lactate and pyruvate output is rarely greater than twofold. This preferential incorporation of pyruvate carbon atoms into fatty acids compared with their release led to the suggestion that insulin might specifically activate the conversion of pyruvate carbon atoms into fatty acid in addition to its well-established effect on glucose uptake (Denton et al., 1968; Denton & Martin, 1970). Subsequently, more clear-cut evidence that insulin accelerates the conversion of pyruvate into fatty acid in fat-pads has been obtained. Thus it has been shown that increases in fatty acid synthesis from glycogen in pads from fasted-re-fed animals or from fructose in pads from fed animals is 1974
