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Biological activity of bovine milk
Livestock Production Science 70 (2001) 79–85 www.elsevier.com / locate / livprodsci
Biological activity of bovine milk Contribution of IGF-I and IGF binding proteins K. Sejrsen*, L.O. Pedersen, M. Vestergaard, S. Purup Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Foulum, PO Box 50, DK-8830 Tjele, Denmark
Abstract The mitogenic activity of milk from different stages of lactation was studied in primary cultures of undifferentiated bovine mammary epithelial cells. The mitogenic activity, measured as DNA synthesis, was 3–4-fold higher in colostrum than in basal medium. The mitogenic activity declined rapidly after calving, and in mid and late lactation the effect was inhibitory, not stimulatory. The content of IGF-I in milk varied with stage of lactation declining from more than 300 ng per ml in colostrum to 1–2 ng per ml in mid lactation and increasing to 20 ng per ml in late lactation. The difference in mitogenic activities between colostrum and mature milk was closely related to the difference in IGF-I content. The changes in IGF-I, however, cannot account for the inhibitory effect of mid and late lactation milk and the difference in the mitogenic effect of colostrum and BPMS (bovine prepartum milk-like secretion). The amounts of IGF binding proteins (24, 28, 34 and 41–44 kDa) in milk followed the same pattern as IGF-I with high content in early lactation and low content in mid lactation. The discrepancies between mitogenic activity and IGF-I content may in some, but not all, cases be related to the content of binding proteins. 2001 Elsevier Science B.V. All rights reserved. Keywords: Bovine milk; IGF-I; IGF binding proteins
1. Introduction Lactation is an integrated part of the reproductive cycle of mammals contributing to the survival, growth and development of the offspring. Milk and colostrum supplies the neonate with nutrients and passive immunity and a large number of biologically active components. The exact biological roles of the various bioactive components in milk are not well documented, but potential biological functions in*Corresponding author. Tel.: 1 45-8999-1513; fax: 1 458999-1525. E-mail address: [email protected] (K. Sejrsen).
clude effects of relevance for the developing neonate as well as auto / paracrine effects in the mammary gland of the mother (Grosvenor et al., 1993). The bioactive components in milk include a large number of hormones and growth factors. Their presence in milk was first described more than 50 years ago (Koldovsky, 1996). Since then the list of hormones and growth factors known to be present in milk and colostrum has grown extensively (Grosvenor et al., 1993) and the list is still growing (Lametsch et al., 2000). Many of the growth factors identified in milk are known to stimulate cell proliferation. This is in line with the fact that both human and bovine milk has
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mitogenic activity in a number of different cell types (Klagburn and Shing, 1984). The relative importance of the various growth factors for the mitogenic activity of milk is not known, but IGF-I is likely to be a major contributor. IGF-I is present in both human and bovine milk in relatively high concentrations — especially in colostrum (Baxter et al., 1984; Malven et al., 1987; Ronge and Blum, 1988). The biological effect of IGF-I is modulated by its binding to IGF binding proteins (Jones and Clemmons, 1995), — of which four have been identified in bovine milk (Campbell and Baumrucker, 1989). The net effect of milk IGF-I therefore is likely to depend on the milk content of the IGFBPs. In this paper we discuss the contribution of IGF-I and IGF binding proteins to the biological effect of milk from different stages of lactation. The discussion is based mainly on results of own investigations and we refer to reviews by Prosser (1996) and Baumrucker and Erondu (2000).
2. Mitogenic activity of milk from different stages of lactation Klagburn and co-workers were the first to demonstrate mitogenic activity of human and bovine milk (Klagburn and Shing, 1984). Their initial observations (Klagburn, 1978) were based on an assay with fibroblasts, but they have demonstrated mitogenic effect of milk in many different cell types. We have developed a bioassay based on primary culture of undifferentiated bovine mammary cells in serum-free medium (Purup et al., 2000a). The cells are cultured in a three dimensional collagen gel for 4–5 days with tritiated thymidine added the last 24 h. We have verified that the DNA synthesis represents epithelial cell proliferation (Ellis et al., 2000; Purup et al., unpublished). With the bioassay we have tested the mitogenic effects of a large number of different hormones and growth factors, including growth hormone, IGF-I, IGF-II, des IGF-I and insulin. The obtained results were in line with the biological actions of the individual growth factors (Purup et al., 2000a,b). The truncated form of IGF-I, des IGF-I, stimulated cell proliferation more effectively than IGF-I, that in turn was more effective than IGF-II, that again was more effective than insulin. As
expected growth hormone had no stimulatory effect. In agreement with previous findings, the effects of IGF-I and IGF-II, but not des-IGF-I, was inhibited by IGFBP-3 (Purup et al., 2000a). The bioassay also is able to detect changes in the mitogenic effect of serum and mammary extracts caused by in vivo treatments such as different feeding levels (Sejrsen et al., 2000; Purup et al., 2000b), stage of development (Norup et al., 1997) and exogenous growth hormone (Sejrsen et al., 1999). Using this bioassay we tested the mitogenic effect of acidified (pH 4.6) and neutralised whey fractions from milk collected from 64 individual cows representing the following 8 stages of lactation: days 1, 2–3, 6–7 and weeks 12 and 24 after parturition, and 1 week before and 1 week after drying off and 3–4 weeks before expected calving (Pedersen, 2000). Adding increasing amounts of whey from colostrum (2–10%) to cell cultures resulted in a 3–4-fold increase in DNA synthesis above basal medium (Fig. 1a). The mitogenic effect of the colostrum / milk, however, dropped rapidly within the first week after calving. In fact by week 12 and onwards, addition of whey to cell cultures medium caused an inhibition of DNA synthesis. By week 24 and 1 week before drying off inhibition was as high as 25–30% by addition of 8–10% milk (Fig. 1b). Interestingly, we observed that addition of low amounts (0.5–1%) of milk-like secretion obtained from dry cows inhibited DNA synthesis, whereas addition of higher amounts (2–8%) stimulated DNA synthesis (Fig. 1c). However, whey obtained from dry cows was much less mitogenic than whey from colostrum. The observed rapid fall in mitogenic activity within the first week of calving is in agreement with earlier findings, but the fall in activity seems to be more rapid and severe in bovine than in human milk (Klagburn and Shing, 1984). Klagburn and Shing observed no mitogenic activity of bovine milk collected 10 days after parturition. They did not observe an inhibitory effect, but they only tested milk from cows up to 10 days after calving. In contrast, they found that the mitogenic activity of human milk was maintained at least 60 days after birth. For the mitogenic effect observed in mammary cells to be of relevance for the neonate, at least two prerequisites have to be fulfilled. Firstly, the growth
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factors involved in the response in mammary cells must also be important for intestinal cells. Although we assume that the effects observed in the mammary cells reflects the variation in mitogenic activity in general, we realise that effects of growth factors vary considerably between tissues. We therefore intend to verify this assumption in an intestinal cell line. Initially we plan to use a cell line developed by Ichiba et al. (1992). They have documented that the proliferation of this cell line is sensitive to human milk as well as to IGF-I and EGF.
3. Contribution of IGF-I to the mitogenic activity of milk
Fig. 1. Effect of adding increasing amounts of neutralised whey from colostrum (day 1) (a), milk from week 24 of lactation (b) or bovine prepartum milk-like secretion (BPMS) collected 3–4 weeks before calving (c) to basal media on 3 H-thymidine incorporation in primary cultures of undifferentiated mammary epithelial cells. Each value is based on the mean of milk from eight cows.
factors responsible for the activity have to maintain activity when they reach the potential target tissue in the intestine. This seems to be the case at least for several growth factors, including IGF-I (Prosser, 1996). Secondly, the biological activity of growth
The IGF-I content of the whey fractions was analysed using a time-resolved immuno-fluoroscens assay (TR-IMFA) developed by Frystyk et al. (1994) and validated for cow serum and milk samples (Vestergaard et al., unpublished). In agreement with previous investigations (Malven et al., 1987; Prosser, 1989; Campbell and Baumrucker, 1988; Schams and Einspanier, 1991) we observed a rapid fall in IGF-I concentration the first week after calving (Pedersen, 2000). In colostrum the average IGF-I concentration was close to 300 ng per ml, and the content dropped to 7 ng per ml 1 week after calving (Fig. 2). This suggests that the reduction in IGF-I concentration is responsible for the drop in mitogenic activity the first week after calving. The involvement of IGF-I is supported by a comparison of the mitogenic effect of IGF-I added to basal medium and the mitogenic effect of colostrum plotted against the amount of IGF-I detected in colostrum (Fig. 3). Below 12.5 ng per ml the two curves mirror each other almost completely. The lack of effect at higher levels is likely to be a result of the IGF-I receptors being gradually saturated and inhibitory effects of other factors such as TGF-b (Purup et al., 2000b), FGFbinding protein (Purup et al., unpublished) or maybe IGF-binding proteins. From week 1 postpartum to mid lactation the IGF-I concentration of milk dropped further to below 2 ng / ml. This is in line with the absence of mitogenic activity in milk from mid lactation, but it is unlikely that the IGF-I content is responsible for the inhibitory effect of whey from mid lactation
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Fig. 2. Mitogenic effect and IGF-I concentration of whey from BPMS, colostrum (day 1) and milk from days 2–3 and days 6–7 after calving. (see text in Fig. 1).
mitogenic effect was low especially compared to colostrum (Fig. 2). This, once again, underlines that the net biological activity is likely to involve many different bioactive components, including IGF binding proteins.
4. Contribution of IGFBPs
Fig. 3. Mitogenic effect of IGF in colostrum (milk day 1) and IGF-I added to basal medium.
milk. The inhibition more likely is due to effects of other factors as mentioned above. From mid lactation to late lactation the IGF-I concentration increased to an average level of 19 ng per ml observed 1 week before drying off. In spite of this increase in IGF-I the whey still caused an inhibition of cell proliferation. In milk collected 1 week after drying off the content was 66 ng per ml and the milk-like secretion collected in the middle of the dry period, 3–4 weeks before calving, contained 4–500 ng / ml in average. In one cow the IGF-I concentration was as high as 1200 ng per ml. In spite of the high concentration in this material, called BPMS (bovine prepartum milklike secretion) (Sandowsky et al., 1993), its
The IGFBPs of milk were evaluated by Western Ligand Blotting essentially according to Hossenlopp et al. (1986). The individual bands were semi-quantified by densitometry and expressed in arbitrary units. We detected four different bands — 24, 28, 34 and 41–44 kDa. According to Gibson et al. (1999) they represent IGFBP-4, -5, -2 and -3, respectively and will be referred to as such. The amounts of binding proteins in milk largely followed the same pattern as IGF-I with high content in BPMS, colostrum and late lactation and low content in milk from mid lactation (Fig. 4). This agrees with earlier findings (Vega et al., 1991; Gibson et al., 1999). We also noted that all IGFBPs, but especially IGFBP-4 and 5 increased rapidly after drying off. This is in line with a possible role of BP-5 in blocking the cell maintenance effect of IGF-I during involution as suggested by Flint and co-workers (Tonner et al., 2000). IGFBP-3 was by far the most abundant binding protein at all stages of lactation, but the relative content of the different binding proteins changed with stage of lactation (Fig. 5). In early lactation the
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Fig. 5. The relative contribution of the different binding proteins to the total IGF-I binding capacity of milk collected at various stages of the lactation cycle (n 5 8).
Fig. 4. IGF binding proteins in milk from different stages of the lactation cycle. Each value is the mean result of eight cows.
relative content of IGFBP-3 dropped to as low as 30%, whereas the relative content of the other binding proteins, especially IGFBP-2, increased. This is most likely a consequence of the negative energy balance at this stage of lactation. It has been observed previously that IGFBP-2 increases when energy balance is low (Vestergaard et al., 1995). In view of the biological actions ascribed to the IGF binding proteins it is relevant to suggest that the observed changes in their content in milk are im-
portant for the biological activity of milk, at least in the mammary gland. In agreement, we have shown that IGFBP-3 inhibits the mitogenic effect of IGF-I, serum and mammary gland extract (Weber et al., 1999). We also observed that the inhibitory effect of IGFBP-3 was higher than the inhibition by antibodies against IGF-I. This suggests that IGFBP-3 also possesses IGF-I independent effects on proliferation of mammary cells. IGFBP-5 also seems to have IGF independent effects (see above). It is therefore possible that the higher content of IGFBPs in BPMS is responsible for the lower mitogenic activity of BPMS compared to colostrum in spite of the higher IGF-I content of BPMS. Another contributing factor to the lower mitogenic effect of BPMS could be, that the relative amount of free IGF-I seems to be much higher in colostrum that in BPMS (Einspanier and Schams, 1991) as is the content of des-IGF-I, which has low affinity to IGFBPs.
5. Concluding remarks The available data support the postulate that colostrum has significant mitogenic effect of potential importance for the mammary gland as well as the developing neonate. The data furthermore suggest that the difference in mitogenic activity of colostrum and mature milk be closely related to changes in IGF-I content. In contrast, differences in IGF-I content cannot explain the difference in
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mitogenic activity of prepartum milk like secretion and colostrum, and it is, similarly, unlikely that IGF-I itself is responsible for the inhibitory effect of late lactation milk on cell proliferation. These discrepancies between IGF-I content and mitogenic activity of the milk may in some, but not all, cases be related to the content of IGF-I binding proteins. It is, however, most likely that the observed mitogenic effect also involve other growth factors and / or proteolytic enzymes capable of modifying the interactions of the various growth factors and binding proteins involved.
References Baumrucker, C.R., Erondu, N.E., 2000. Insulin-like growth factor (IGF) system in the bovine mammary gland and milk. J. Mamm. Gland Biol. Neoplasia 5, 53–64. Baxter, R., Zaltsman, Z., Turtle, J.R., 1984. Immunoreactive somatomedin C / insulin-like growth factor-I and its binding protein in human milk. J. Endocrinol. Metab. 58, 955–959. Campbell, P.G., Baumrucker, C.R., 1989. Insulin-like growth factor-I and insulin-like growth factor binding proteins in bovine milk. J. Endocrinol. 120, 21–29. Einspanier, R., Schams, D., 1991. Changes in concentrations of insulin-like growth factor 1, insulin and growth hormone in bovine mammary gland secretion ante and post partum. J. Dairy Res. 58, 171–178. Ellis, S., Purup, S., Sejrsen, K., Akers, R.M., 2000. Differences in the proliferation and morhpogenesis of mammary organoids from the peripheral and medial parenchymal zones of the prepubertal bovine mammary gland. J. Dairy Sci. 81 (Suppl. 1), 375. Frystyk, J., Skjærbæk, C., Dinesen, B., Ørskov, H., 1994. Free insulin-like growth factors (IGF-I and IGF-II) in human serum. FEBS Lett. 348, 185–191. Gibson, C.A., Staley, M.D., Baumrucker, C.R., 1999. Identification of IGF binding proteins in bovine milk and the demonstration of IGFBP-3 synthesis and release by bovine mammary epithelial cells. J. Anim. Sci. 77, 1547–1557. Grosvenor, C.E., Picciano, M.F., Baumrucker, C.R., 1993. Hormones and growth factors in milk. Endocr. Rev. 14, 710–728. Hossenlopp, P., Seurin, D., Segovia-Quinson, B., Hardouin, S., Binoux, M., 1986. Analysis of serum insulin-like growth factor binding proteins using western ligand blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal. Biochem. 154, 138–143. Ichiba, H., Kusada, S., Itagane, Y., Fujita, K., Issiki, 1992. Measurement of growth promoting activity in human milk using a fetal small intestinal cell line. Biol. Neonate, 61, 47–53. Jones, J.I., Clemmons, D.R., 1995. Insulin-like growth factors and their biological actions. Endocr. Rev. 16, 3–34.
Klagburn, M., 1978. Human milk stimulates DNA synthesis and cellular proliferation in cultured fibroblasts. Proc. Natl. Acad. Sci. USA 75, 5057–5061. Klagburn, M., Shing, Y., 1984. Growth-promoting factors in human and bovine milk. Growth Matur. Factors 2, 161–192. Koldovsky, O., 1996. The potential physiological significance of milk-borne hormonally active substances for the neonate. J. Mamm. Gland Biol. Neoplasia 1, 317–323. Lametsch, R., Rasmussen, J.T., Johnsen, L.B., Purup, S., Sejrsen, K., Petersen, T.E., Heegaard, C.W., 2000. Structural characterisation of the Fibroblast Growth Factor Binding Protein purified from bovine mammary gland secretion. J. Biol. Biochem. 275, 19469–19474. Malven, P.V., Head, H.H., Collier, R.J., Bounomo, F.C., 1987. Periparturient changes in secretion and mammary uptake of insulin and in concentrations if insulin and insulin-like growth factors in milk of dairy cows. J. Dairy Sci. 70, 2254–2265. Norup, L.R., Purup, S., Sejrsen, K., 1997. Mitogenic effect of serum from heifers of different ages on proliferation of mammary epithelial cells in collagen gel culture. 48th Annual Meeting of EAAP, Book of Abstracts, 183. Pedersen, L.O., 2000. Insulin-like growth Factor-I (IGF-I) og IGF-bindingsproteiner i mælk: Betydning for mælkens mitogene effekt. M.Sc. thesis. The Royal Veterinary and Agricultural University, Copenhagen, pp. 72. Prosser, C.G., 1996. Insulin-like growth factors in milk and mammary gland. J. Mamm. Gland Biol. Neoplasia 1, 297–306. Purup, S., Vestergaard, M., Sejrsen, K., 2000a. Involvement of growth factors in the regulation of pubertal mammary growth. Adv. Exp. Med. Biol. 480, 27–43. Purup, S., Vestergaard, M., Weber, M.S., Plaut, K., Akers, R.M., Sejrsen, K., 2000b. Local regulation of pubertal mammary growth in heifers. J. Anim. Sci. 78 (Suppl. 3), 36–47. Ronge, H., Blum, J., 1988. Somatomedin C and other hormones in dairy cows around parturition, in new-born calves and in milk. J. Anim. Physiol. & Anim. Nutr. 60, 168–174. Sandowsky, Y., Peri, I., Gertler, A., 1993. Partial purification and characterization of putative paracrine / autocrine bovine mammary epithelium growth factors. Livest. Prod. Sci. 35, 35–48. Schams, D., Einspanier, R., 1991. Growth hormone, IGF-I and insulin in mammary gland secretion before and after parturition and possibility of their transfer into the calf. Endocr. Regul. 25, 139–143. Sejrsen, K., Purup, S., Vestergaard, M., Weber, M.S., Knight, C.H., 1999. Growth hormone and mammary development. Domest. Anim. Endocrinol. 17, 117–129. Sejrsen, K., Purup, S., Vestergaard, M., Foldager, J., 2000. High body weight gain and reduced bovine mammary growth: physiological basis and implications for potential milk yields. Domest. Anim. Endocrinol. 19, 93–104. Tonner, E., Allan, G., Shkreta, L., Webster, J., Whitelaw, D., Flint, D.J., 2000. Insulin-like growth factor binding protein-5 (IGFBP-5) potentially regulates programmed cell death and plasminogen activation in the mammary gland. Adv. Exp. Med. Biol. 480, 45–54. Vega, J.R., Gibson, C.A., Skaar, T.C., Hadsell, D.L., Baumrucker, C.R., 1991. Insulin-like growth factor (IGF)-I and II and IGF binding proteins in serum and mammary secretions during the
K. Sejrsen et al. / Livestock Production Science 70 (2001) 79 – 85 dry period and early lactation in dairy cows. J. Anim. Sci. 69, 2538–2547. Vestergaard, V., Purup, S., Sejrsen, K., 1995. Influence of bovine growth hormone and feeding level on growth and endocrine status of prepubertal heifers. J. Anim. Sci. 73 (Suppl. 1), 148.
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Weber, M.S., Purup, S., Vestergaard, M., Ellis, S.E., Akers, R.M., Sejrsen, K., 1999. Contribution of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3) to mitogenic activity in bovine mammary extracts and serum. J. Endocrinol. 161, 363–365.
Biological activity of bovine milk Contribution of IGF-I and IGF binding proteins K. Sejrsen*, L.O. Pedersen, M. Vestergaard, S. Purup Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Foulum, PO Box 50, DK-8830 Tjele, Denmark
Abstract The mitogenic activity of milk from different stages of lactation was studied in primary cultures of undifferentiated bovine mammary epithelial cells. The mitogenic activity, measured as DNA synthesis, was 3–4-fold higher in colostrum than in basal medium. The mitogenic activity declined rapidly after calving, and in mid and late lactation the effect was inhibitory, not stimulatory. The content of IGF-I in milk varied with stage of lactation declining from more than 300 ng per ml in colostrum to 1–2 ng per ml in mid lactation and increasing to 20 ng per ml in late lactation. The difference in mitogenic activities between colostrum and mature milk was closely related to the difference in IGF-I content. The changes in IGF-I, however, cannot account for the inhibitory effect of mid and late lactation milk and the difference in the mitogenic effect of colostrum and BPMS (bovine prepartum milk-like secretion). The amounts of IGF binding proteins (24, 28, 34 and 41–44 kDa) in milk followed the same pattern as IGF-I with high content in early lactation and low content in mid lactation. The discrepancies between mitogenic activity and IGF-I content may in some, but not all, cases be related to the content of binding proteins. 2001 Elsevier Science B.V. All rights reserved. Keywords: Bovine milk; IGF-I; IGF binding proteins
1. Introduction Lactation is an integrated part of the reproductive cycle of mammals contributing to the survival, growth and development of the offspring. Milk and colostrum supplies the neonate with nutrients and passive immunity and a large number of biologically active components. The exact biological roles of the various bioactive components in milk are not well documented, but potential biological functions in*Corresponding author. Tel.: 1 45-8999-1513; fax: 1 458999-1525. E-mail address: [email protected] (K. Sejrsen).
clude effects of relevance for the developing neonate as well as auto / paracrine effects in the mammary gland of the mother (Grosvenor et al., 1993). The bioactive components in milk include a large number of hormones and growth factors. Their presence in milk was first described more than 50 years ago (Koldovsky, 1996). Since then the list of hormones and growth factors known to be present in milk and colostrum has grown extensively (Grosvenor et al., 1993) and the list is still growing (Lametsch et al., 2000). Many of the growth factors identified in milk are known to stimulate cell proliferation. This is in line with the fact that both human and bovine milk has
0301-6226 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 01 )00199-3
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mitogenic activity in a number of different cell types (Klagburn and Shing, 1984). The relative importance of the various growth factors for the mitogenic activity of milk is not known, but IGF-I is likely to be a major contributor. IGF-I is present in both human and bovine milk in relatively high concentrations — especially in colostrum (Baxter et al., 1984; Malven et al., 1987; Ronge and Blum, 1988). The biological effect of IGF-I is modulated by its binding to IGF binding proteins (Jones and Clemmons, 1995), — of which four have been identified in bovine milk (Campbell and Baumrucker, 1989). The net effect of milk IGF-I therefore is likely to depend on the milk content of the IGFBPs. In this paper we discuss the contribution of IGF-I and IGF binding proteins to the biological effect of milk from different stages of lactation. The discussion is based mainly on results of own investigations and we refer to reviews by Prosser (1996) and Baumrucker and Erondu (2000).
2. Mitogenic activity of milk from different stages of lactation Klagburn and co-workers were the first to demonstrate mitogenic activity of human and bovine milk (Klagburn and Shing, 1984). Their initial observations (Klagburn, 1978) were based on an assay with fibroblasts, but they have demonstrated mitogenic effect of milk in many different cell types. We have developed a bioassay based on primary culture of undifferentiated bovine mammary cells in serum-free medium (Purup et al., 2000a). The cells are cultured in a three dimensional collagen gel for 4–5 days with tritiated thymidine added the last 24 h. We have verified that the DNA synthesis represents epithelial cell proliferation (Ellis et al., 2000; Purup et al., unpublished). With the bioassay we have tested the mitogenic effects of a large number of different hormones and growth factors, including growth hormone, IGF-I, IGF-II, des IGF-I and insulin. The obtained results were in line with the biological actions of the individual growth factors (Purup et al., 2000a,b). The truncated form of IGF-I, des IGF-I, stimulated cell proliferation more effectively than IGF-I, that in turn was more effective than IGF-II, that again was more effective than insulin. As
expected growth hormone had no stimulatory effect. In agreement with previous findings, the effects of IGF-I and IGF-II, but not des-IGF-I, was inhibited by IGFBP-3 (Purup et al., 2000a). The bioassay also is able to detect changes in the mitogenic effect of serum and mammary extracts caused by in vivo treatments such as different feeding levels (Sejrsen et al., 2000; Purup et al., 2000b), stage of development (Norup et al., 1997) and exogenous growth hormone (Sejrsen et al., 1999). Using this bioassay we tested the mitogenic effect of acidified (pH 4.6) and neutralised whey fractions from milk collected from 64 individual cows representing the following 8 stages of lactation: days 1, 2–3, 6–7 and weeks 12 and 24 after parturition, and 1 week before and 1 week after drying off and 3–4 weeks before expected calving (Pedersen, 2000). Adding increasing amounts of whey from colostrum (2–10%) to cell cultures resulted in a 3–4-fold increase in DNA synthesis above basal medium (Fig. 1a). The mitogenic effect of the colostrum / milk, however, dropped rapidly within the first week after calving. In fact by week 12 and onwards, addition of whey to cell cultures medium caused an inhibition of DNA synthesis. By week 24 and 1 week before drying off inhibition was as high as 25–30% by addition of 8–10% milk (Fig. 1b). Interestingly, we observed that addition of low amounts (0.5–1%) of milk-like secretion obtained from dry cows inhibited DNA synthesis, whereas addition of higher amounts (2–8%) stimulated DNA synthesis (Fig. 1c). However, whey obtained from dry cows was much less mitogenic than whey from colostrum. The observed rapid fall in mitogenic activity within the first week of calving is in agreement with earlier findings, but the fall in activity seems to be more rapid and severe in bovine than in human milk (Klagburn and Shing, 1984). Klagburn and Shing observed no mitogenic activity of bovine milk collected 10 days after parturition. They did not observe an inhibitory effect, but they only tested milk from cows up to 10 days after calving. In contrast, they found that the mitogenic activity of human milk was maintained at least 60 days after birth. For the mitogenic effect observed in mammary cells to be of relevance for the neonate, at least two prerequisites have to be fulfilled. Firstly, the growth
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factors involved in the response in mammary cells must also be important for intestinal cells. Although we assume that the effects observed in the mammary cells reflects the variation in mitogenic activity in general, we realise that effects of growth factors vary considerably between tissues. We therefore intend to verify this assumption in an intestinal cell line. Initially we plan to use a cell line developed by Ichiba et al. (1992). They have documented that the proliferation of this cell line is sensitive to human milk as well as to IGF-I and EGF.
3. Contribution of IGF-I to the mitogenic activity of milk
Fig. 1. Effect of adding increasing amounts of neutralised whey from colostrum (day 1) (a), milk from week 24 of lactation (b) or bovine prepartum milk-like secretion (BPMS) collected 3–4 weeks before calving (c) to basal media on 3 H-thymidine incorporation in primary cultures of undifferentiated mammary epithelial cells. Each value is based on the mean of milk from eight cows.
factors responsible for the activity have to maintain activity when they reach the potential target tissue in the intestine. This seems to be the case at least for several growth factors, including IGF-I (Prosser, 1996). Secondly, the biological activity of growth
The IGF-I content of the whey fractions was analysed using a time-resolved immuno-fluoroscens assay (TR-IMFA) developed by Frystyk et al. (1994) and validated for cow serum and milk samples (Vestergaard et al., unpublished). In agreement with previous investigations (Malven et al., 1987; Prosser, 1989; Campbell and Baumrucker, 1988; Schams and Einspanier, 1991) we observed a rapid fall in IGF-I concentration the first week after calving (Pedersen, 2000). In colostrum the average IGF-I concentration was close to 300 ng per ml, and the content dropped to 7 ng per ml 1 week after calving (Fig. 2). This suggests that the reduction in IGF-I concentration is responsible for the drop in mitogenic activity the first week after calving. The involvement of IGF-I is supported by a comparison of the mitogenic effect of IGF-I added to basal medium and the mitogenic effect of colostrum plotted against the amount of IGF-I detected in colostrum (Fig. 3). Below 12.5 ng per ml the two curves mirror each other almost completely. The lack of effect at higher levels is likely to be a result of the IGF-I receptors being gradually saturated and inhibitory effects of other factors such as TGF-b (Purup et al., 2000b), FGFbinding protein (Purup et al., unpublished) or maybe IGF-binding proteins. From week 1 postpartum to mid lactation the IGF-I concentration of milk dropped further to below 2 ng / ml. This is in line with the absence of mitogenic activity in milk from mid lactation, but it is unlikely that the IGF-I content is responsible for the inhibitory effect of whey from mid lactation
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Fig. 2. Mitogenic effect and IGF-I concentration of whey from BPMS, colostrum (day 1) and milk from days 2–3 and days 6–7 after calving. (see text in Fig. 1).
mitogenic effect was low especially compared to colostrum (Fig. 2). This, once again, underlines that the net biological activity is likely to involve many different bioactive components, including IGF binding proteins.
4. Contribution of IGFBPs
Fig. 3. Mitogenic effect of IGF in colostrum (milk day 1) and IGF-I added to basal medium.
milk. The inhibition more likely is due to effects of other factors as mentioned above. From mid lactation to late lactation the IGF-I concentration increased to an average level of 19 ng per ml observed 1 week before drying off. In spite of this increase in IGF-I the whey still caused an inhibition of cell proliferation. In milk collected 1 week after drying off the content was 66 ng per ml and the milk-like secretion collected in the middle of the dry period, 3–4 weeks before calving, contained 4–500 ng / ml in average. In one cow the IGF-I concentration was as high as 1200 ng per ml. In spite of the high concentration in this material, called BPMS (bovine prepartum milklike secretion) (Sandowsky et al., 1993), its
The IGFBPs of milk were evaluated by Western Ligand Blotting essentially according to Hossenlopp et al. (1986). The individual bands were semi-quantified by densitometry and expressed in arbitrary units. We detected four different bands — 24, 28, 34 and 41–44 kDa. According to Gibson et al. (1999) they represent IGFBP-4, -5, -2 and -3, respectively and will be referred to as such. The amounts of binding proteins in milk largely followed the same pattern as IGF-I with high content in BPMS, colostrum and late lactation and low content in milk from mid lactation (Fig. 4). This agrees with earlier findings (Vega et al., 1991; Gibson et al., 1999). We also noted that all IGFBPs, but especially IGFBP-4 and 5 increased rapidly after drying off. This is in line with a possible role of BP-5 in blocking the cell maintenance effect of IGF-I during involution as suggested by Flint and co-workers (Tonner et al., 2000). IGFBP-3 was by far the most abundant binding protein at all stages of lactation, but the relative content of the different binding proteins changed with stage of lactation (Fig. 5). In early lactation the
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Fig. 5. The relative contribution of the different binding proteins to the total IGF-I binding capacity of milk collected at various stages of the lactation cycle (n 5 8).
Fig. 4. IGF binding proteins in milk from different stages of the lactation cycle. Each value is the mean result of eight cows.
relative content of IGFBP-3 dropped to as low as 30%, whereas the relative content of the other binding proteins, especially IGFBP-2, increased. This is most likely a consequence of the negative energy balance at this stage of lactation. It has been observed previously that IGFBP-2 increases when energy balance is low (Vestergaard et al., 1995). In view of the biological actions ascribed to the IGF binding proteins it is relevant to suggest that the observed changes in their content in milk are im-
portant for the biological activity of milk, at least in the mammary gland. In agreement, we have shown that IGFBP-3 inhibits the mitogenic effect of IGF-I, serum and mammary gland extract (Weber et al., 1999). We also observed that the inhibitory effect of IGFBP-3 was higher than the inhibition by antibodies against IGF-I. This suggests that IGFBP-3 also possesses IGF-I independent effects on proliferation of mammary cells. IGFBP-5 also seems to have IGF independent effects (see above). It is therefore possible that the higher content of IGFBPs in BPMS is responsible for the lower mitogenic activity of BPMS compared to colostrum in spite of the higher IGF-I content of BPMS. Another contributing factor to the lower mitogenic effect of BPMS could be, that the relative amount of free IGF-I seems to be much higher in colostrum that in BPMS (Einspanier and Schams, 1991) as is the content of des-IGF-I, which has low affinity to IGFBPs.
5. Concluding remarks The available data support the postulate that colostrum has significant mitogenic effect of potential importance for the mammary gland as well as the developing neonate. The data furthermore suggest that the difference in mitogenic activity of colostrum and mature milk be closely related to changes in IGF-I content. In contrast, differences in IGF-I content cannot explain the difference in
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mitogenic activity of prepartum milk like secretion and colostrum, and it is, similarly, unlikely that IGF-I itself is responsible for the inhibitory effect of late lactation milk on cell proliferation. These discrepancies between IGF-I content and mitogenic activity of the milk may in some, but not all, cases be related to the content of IGF-I binding proteins. It is, however, most likely that the observed mitogenic effect also involve other growth factors and / or proteolytic enzymes capable of modifying the interactions of the various growth factors and binding proteins involved.
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