0013-7227/90/1261-0637$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Characterization of the Change in Type I and II InsulinLike Growth Factor Receptors of Bovine Mammary Tissue during the Pre- and Postpartum Periods* DARRYL L. HADSELL, PHIL G. CAMPBELL, AND CRAIG R. BAUMRUCKER Department of Dairy and Animal Science, Pennsylvania State University (D.L.H., C.R.B.), University Park, Pennsylvania 16802; and the Allegheny-Singer Research Institute ((P.G.C.), Pittsburgh, Pennsylvania 15212-9986
ABSTRACT. Type I and II receptors for the insulin-like growth factors (IGF) were characterized in microsomes from bovine mammary tissue. Specific binding of [125I]IGF-I increased linearly with increasing microsomal protein concentrations. In contrast, IGF-II binding showed a curvilinear relationship over the concentration range tested, with a maximum at 120 ng/va\. Kinetic studies conducted at 4 C showed the binding reactions to be reversible. Competition studies showed recombinant human IGF-I (rh-IGF-I) to be 8-, 18-, and 1000-fold more potent at inhibiting [125I]rh-IGF-I binding than recombinant bovine IGF-II (rb-IGF-II), multiplication-stimulating activity, and insulin, respectively. rbIGF-II was 1.8- and 6-fold more potent at inhibiting [125I]rbIGF-II binding than rhIGF-II and multiplication-stimulating activity. Specific binding of [125I]IGF-I and -II
was measured in microsomes prepared from cows (n = 47) ranging from 138 days prepartum to 411 days postpartum. IGFI binding declined during the prepartum period, increased 75% with the onset of lactation, and then declined during the postpartum period. Scatchard analysis showed the presence of a single class of high affinity binding sites for both ligands, with type II receptors being about 10-fold more prevalent than type I receptors. The survey and Scatchard data support the conclusion that the onset of lactation coincides with an increase in the number of type I receptors present in mammary tissue. This increase supports the contention that IGF-I may play an important role in modulating the metabolic activity of the bovine mammary gland. {Endocrinology 126: 637-643, 1990)
R
(4). Further, lactogenesis in the cow is associated with increased binding of IGF-I to mammary microsomes (5). In contrast, type I receptors are higher during gestation for the rat and ewe than they are during lactation (2, 6). The type II receptors are higher in pregnant and lactating rats than in virgin rats, with no apparent change in type II receptors with the onset of lactation. In the ewe the type II receptors are lower during lactation than they are during the prepartum period. These findings, while varied across species, suggest that the IGF receptors play an important role in modulation of the growth and secretory function of the mammary gland. The objectives of this study were to characterize in greater detail the receptors for IGFs in the microsomal fraction of bovine mammary tissue. The focus of these studies was on specificity, kinetic properties, affinity, and binding capacity of the IGF receptors in the mammary gland during the prepartum (nonlactating) and postpartum (lactating) periods.
ECEPTORS for insulin-like growth factors (IGFs) have been demonstrated in mammary tissue from rabbit (1), rat (2), pig (3), human (4), cow (5), and ewe (6). In the cow these receptors are currently believed to be involved in the IGF-mediated actions of GH on the mammary gland (5). This belief is based on the failure to detect receptors for GH in bovine mammary tissue (7), and the failure of GH to stimulate growth of or milk secretion by bovine mammary tissue in vitro (8). The mechanism by which IGFs could mediate the GH response in mammary tissue is analogous to a hypothesis that was initially proposed to describe stimulation of bone growth by GH (9). Mammary gland IGF receptors have been reported to change with the physiological state of the tissue. For example, transformation of human mammary tissue to a malignant state coincides with increased IGF-I binding Received August 21, 1989. Address requests for reprints to: Dr. Craig R. Baumrucker, Department of Dairy and Aninial Science, Pennsylvania State University, 302 Henning Building, University Park, Pennsylvania 16802. * This work was supported by the Pennsylvania Agricultural Experimental Station and USDA Grant 85-CRCR-1-1881. Authorization for publication as paper 8278 in the journal series of the Pennsylvania Agricultural Experimental Station.
Materials and Methods Materials Recombinant bovine IGF-II (rb-IGF-II) was a generous gift of Dr. Robert Collier (Monsanto Corp., St. Louis, MO). Recom637
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IGF RECEPTORS IN MAMMARY TISSUE
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binant human IGF-I (rh-IGF-I) was a generous gift of Dr. Dan Burleigh (Pitman-Moore, Inc., Terre Haute, IN). rh-IGF-II was purchased from Bachem, Inc. (Torrence, CA). Na125I was obtained from Amersham International (Arlington Heights, IL). Porcine proinsulin was obtained from Eli Lily Co. (Indianapolis, IN). Bovine GH (bGH) and bovine PRL (bPRL) were gifts from the NIDKK. All other reagents were of analytical grade. Preparation of microsomes Mammary tissue was collected from prepartum and postpartum dairy cows that were slaughtered by the Pennsylvania State University Dairy Research Facility for reasons other than mastitis. All cows in this study had one or more lactations. The tissue was placed on ice for transport from the slaughter facility to the laboratory and then frozen at -20 C until preparation of microsomes. Microsomes were prepared as described by Pocius et al. (10) and then stored at -80 C. The protein concentration of the microsome preparations was determined by the bicinchoninic acid assay (Pierce, Rockford, IL). IGFRRA IGF receptor-binding activity was measured using a modification of the insulin RRA of Oscar et al. (11). IGF-I and -II were iodinated to a specific activity of 100-300 ^Ci/jug, using chloramine-T and purified by the method of Etherton et al. (12). The assay was performed using 12 X 75-mm polypropylene tubes in 750 n\ 50 mM Tris butfer (pH 7.8) containing 0.5% BSA. Microsomal protein was incubated with 0.2 ng/ml [125I] IGF-I or -II for 48 h at 4 C unless otherwise stated. Receptorbound hormone was separated from free by centrifugation at 3000 x g at 4 C for 30 min. The pellet was counted in a model 1274 RIAGAMMA counter (LKB Instruments, Rockville, MD). Nonspecific binding was determined in the presence of 333 ng/ml unlabeled IGF-I or -II.
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Data analysis Scatchard plots for IGF-I and -II binding were fit by least squares linear regression. The kinetic and inhibition data were analyzed with the Graphpad data analysis software package (ISI Software, Philadelphia, PA). Analysis of the survey data was performed by discontinuous least squares linear regression. The Scatchard parameter estimates from pre- and postpartum cows were analyzed by a one-way analysis of variance (13).
Results Protein dependence Both total and specific binding of [125I] IGF-I (Fig. 1A) increased in a linear fashion over the range of microsomal protein tested. From this study 667 /u.g/ml microsomal protein were selected for all subsequent IGF-I RRAs. At this concentration of microsomal protein nonspecific binding was about 30% of total binding. Total binding of [125I]IGF-II as a function of microsomal protein concentration (Fig. IB) increased in a hyperbolic fashion (y = 0.27[1 - e0017x] + 0.148; P < 0.005), approaching an asymptotic maximum of 41% of the total counts added at 667 fig/ml membrane protein. Nonspecific binding increased in a linear fashion (r2 = 0.92). Specific binding as a function of microsomal protein was best fit by a mixed exponential function (y = 0.07[e-°007x] + 0.26[l - e"003*] - 0.0004; P < 0.005), showing a maximum at 120 ng/m\. At this dose of micro0.15-,
A
0.12 0.09
Receptor characterization
0.06
The protein dependence of [125I]IGF binding was assayed by testing 133-880 Mg/ml microsomal protein for rhIGF-I and 9667 Mg/ml for rbIGF-II. The time dependence of association was determined at 4 C with 667 Mg/ml microsomal protein for rhIGF-I and 133 Mg/ml microsomal protein for rbIGF-II. The dissociation reaction was followed for 96 h after adding a 1700fold molar excess IGF-I or -II to microsomes that were preequilibrated with [125I]IGF-I or -II for 48 h. The specificity of [125I]rhIGF-I and [125I]rbIGF-II binding was measured in the presence of varying concentrations of rhIGF-I, rbIGF-II, rhIGF-II, bovine insulin, multiplication-stimulating activity (MSA), porcine proinsulin, bGH, and bPRL. A survey of specific binding of [125I]rhIGF-I or [125I]rbIGF-II was carried out with microsomes from 46 individual cows ranging from -138 to 411 days relative to parturition. The concentration of microsomal protein for both IGF-I and -II was 667 Mg/ml. Scatchard analysis was performed on the binding of rhIGF-I and rhlGFII from 5 prepartum and 5 postpartum cows using 667 Mg/ml microsomal protein for IGF-I and 133 Mg/ml microsomal protein for IGF-II. The concentration of unlabeled IGF-I or -II in both assays ranged from 0-33 ng/ml.
0.03 ^ 0.00 200
400
600
800
MICROSOMAL PROTEIN. (jiq/m\)
0.40 0.300.20 0.10
0.00 100
200 300 400 500 600 700 MICROSOMAL PROTEIN, (jtq/m\)
800
FIG. 1. Total (O), specific (0), and nonspecific (A) binding of [125I] rhIGF-I (A) and [125I]rbIGF-II (B) as a function of mammary microsome concentration. Each point represents the mean ± SEM for three cows. Various concentrations of microsomal protein were incubated with 0.2 ng/ml [125I]rhIGF-I or [125I]rbIGF-II in a total volume of 0.75 ml for 48 h at 4 C. Nonspecific binding was measured in the presence of a 1700-fold molar excess of IGF-I or -II. B/T, Bound to total ratio.
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IGF RECEPTORS IN MAMMARY TISSUE somal protein, the nonspecific binding of IGF-II was about 25% of the total binding. Unless otherwise stated, 133 Mg/ml microsomal protein was chosen for all IGF-II RRAs.
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g 60.0a. l" 40.0E
Association/dissociation [125I]IGF-I associated (Fig. 2A) to the membranes with a rate constant of 0.26 fmol/mg protein-h, approaching an asymptotic maximum of 1.86 fmol/mg protein. Equilibrium was reached for this reaction within 24 h. The rate of [125I]IGF-I dissociation (Fig. 2B) was 0.06 fmol/ mg protein-h. About 30% of the [125I]IGF-I bound at equilibrium remained bound 96 h after the addition of excess unlabeled IGF-I. [125I]IGF-II binding increased (Fig. 3A) with a rate constant of 0.14 fmol/mg proteinh and reached an asymptotic maximum of 44.61 fmol/ mg protein. Equilibrium was reached by about 30 h. The dissociation of [125I]IGF-II (Fig. 3B) proceeded with a rate constant of 0.03 fmol/mg protein-h. About 42% of 3.01
J I
FiG. 3. Association (A) and dissociation (B) kinetics for the binding of [125I]rbIGF-II to mammary microsomes. Each point represents the mean ± SEM for two cows. A, 133 Mg/ml microsomal protein were incubated with 0.2 ng/ml [125I]rbIGF-II for various times at 4 C in a total volume of 0.75 ml. B, 133 Mg/ml microsomal protein were equilibrated for 48 h with 0.2 ng/ml [125I]rbIGF-II in a total volume of 0.75 ml. After the equilibration step, a 1700-fold molar excess of rblGF-II was added to each tube, and binding was measured at various times.
the [125I] IGF-II bound at equilibrium remained bound to the membranes by 96 h.
I I
20
TIME, (h)
Specificity
60
40
80
TIME, (h) 2.0 n
40
60
100
TIME, (h)
FIG. 2. Association (A) and dissociation (B) kinetics for the binding of [126I]rhIGF-I to mammary microsomes. Each point represents the mean ± SEM for two cows. A, 667 Mg/ml microsomal protein in a total volume of 0.75 ml were incubated with 0.2 ng/ml [125I]rhIGF-I for various times at 4 C. B, 667 Mg/ml microsomal protein were equilibrated for 48 h with 0.2 ng/ml [125I]rhIGF-I in a total volume of 0.75 ml. After the equilibration step, a 1700-fold molar excess of rhIGF-I was added to each tube, and binding was measured at various times.
Specific binding of [125I]rhIGF-I (Fig. 4A) in the presence of 0-667 ng/ml rhIGF-I, rblGF-II, or MSA showed ED50 values of 3.6 ± 1.4, 30.1 ± 1.5, and 67.3 ± 15 ng/ ml, respectively. The ED50 of insulin for inhibition of [125I]rhIGF-I binding was 3.6 ± 0.002 Mg/ml. Proinsulin could only inhibit about 25% of [125I]rhIGF-I binding at a concentration of 6.7 jug/ml. Neither bGH nor bPRL could inhibit [125I]rhIGF-I binding at concentrations of up to 13 Mg/ml (data not shown). Specific binding of [125I]rbIGF-II (Fig. 4B) was measured with 667 fig/ml microsomal protein in the presence of 0-133 ng/ml rblGF-II or 0-267 ng/ml MSA. The ED50 values for rblGF-II and MSA were 13.6 ± 1.4 and 82.34 ± 1 . 1 ng/ml, respectively. rhIGF-I, insulin, proinsulin, GH, and PRL failed to inhibit [125I]rbIGF-II binding at concentrations of up to 0.67, 67, 6.60, 13, and 13 ng/ml, respectively (data shown for rhIGF-I only). In a separate IGF-II RRA, the potency of rhIGF-II was compared with that of rblGF-II using 133 Mg/ml microsomal protein and [125I]rbIGF-II The ED50 for rblGF-II in this case was 13.2 ± 1.1 ng/ml, while that for rhIGF-II was 25.1 ± 1.0 ng/ml (Fig. 5). Pre- and postpartum IGF-binding activity Binding of [125I]IGF-I and -II as a function of physiological state and days relative to parturition is presented
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IGF RECEPTORS IN MAMMARY TISSUE
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Endo • 1990 Voll26«Nol
1.25T
1.00 0.75 0.50 0.25
1.0E-2
0.1
1.0
10.0
100.0 1000.0 1.0E4
1.0E5
COMPETITOR, (ng/ml)
B
1.00
*> -c^uur p " ° ° o o o £^JTUF o
0.75-
-150-100 -50
0
0.50-
\
50
100 150 200 250 300 350 400 450
TIME, (days postpartum)
\
0.25
\ 0.00
1.0E-2
0.1
^
100-,
1.0 10.0 100.0 1000.0 1.0E4 COMPETITOR, (ng/ml)
1.0E5
FIG. 4. Inhibition of [125I]rhIGF-I (A) and [12SI]rbIGF-II (B) binding to pooled mammary microsomes (five nonlactating and five lactating cows) by rhIGF-I (O), rbIGF-II (A), MSA (•), bovine insulin (V), and porcine proinsulin (0). A, 667 fig/ml microsomal protein were incubated with 0.2 ng/ml [125I]rhIGF-I and various concentrations of the above competitors (see Materials and Methods) for 48 h at 4 C in a total volume of 0.75 ml. B, 667 jig/ml microsomal protein were incubated with 0.2 ng/ml [125I]rbIGF-II and various concentrations of competitors for 48 h at 4 C in a total volume of 0.75 ml. B/BO, Ratio of bound in the presence of competitor to bound in the absence of competitor.
«
80
e £ 60\ "5 O .E 40
O.
oo
20-
-150-100 -50 1.25y
o o
O
0
50
100 150 200 250 300 350 400 450
TIME, (days postpartum)
FIG. 6. Specific binding of [125I]rhIGF-I (A) and [126I]rbIGF-II (B) to mammary microsomes as a function of physiological state (nonlactating vs. lactating) and days relative to parturition. Each point represents the binding to a microsome preparation from one cow. Binding was measured after incubation of 667 Mg/ml microsomal protein with 0.2 ng/ml [125I]rhIGF-I or [125I]rbIGF-II for 48 h at 4 C in a total volume of 0.75 ml.
1.00-
0.75-
0.50-
1
10
100
1000
COMPETITOR, (ng/ml)
125
FIG. 5. Inhibition of [ I]rbIGF-I binding to pooled mammary nucrosomes (five nonlactating and five lactating cows) by rblGF-II-(A) and rhIGF-II (0). A 48-h incubation of 0.2 ng/ml [125I]rbIGF-II with 133 jig/ml microsomal protein and various concentrations of rbIGF-II or rhIGF-II (see Materials and Methods) was conducted at 4 C. B/BO, Bound to free ratio.
in Fig. 6. The data were analyzed by least squares linear regression. Regression of bound [125I]IGF-I on days relative to partuntion resulted in a significant fit of the model (P < 0.01). Both the intercept and the slope of the regression line for the postpartum group were different from those for the prepartum group. These data show that IGF-I binding decreased during the prepartum period, increased with the onset of lactation, and then decreased during the postpartum period.. IGF-I binding in the prepartum group declined at a rate of 0.025 fmol/ mg protein • day and intersected the y-axis at 3.07 fmol/ mg protein-day. Binding in the postpartum group declined by 0.004 fmol/mg protein • day and intersected the
y-axis at 5.4 fmol/mg protein. [125I]IGF-II binding appeared to follow the same pattern as IGF-I binding. However, due to larger cow to cow variation and an apparently smaller difference between the pre- and postpartum groups, no change in IGF-II binding was detected with the onset of lactation or with time during the preor postpartum period (Fig. 6B). Scatchard analysis of pre- and postpartum binding A comparison of the estimated Kd and binding capacity for [125I]IGF binding was obtained by Scatchard analysis of the binding data from five prepartum (-40 ± 18 days) and five postpartum (21.6 ± 12.4 days) cows (Fig. 7). Both receptors behaved as a single class of high affinity sites. The affinity and binding capacity of the IGF receptors are presented in Table 1. The analysis of variance failed to detect a significant difference in affinity or binding capacity for either ligand between the pre- and
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IGF RECEPTORS IN MAMMARY TISSUE 0.12 0.10 0.08 0.060.04 0.02 0.00 0.05
0.10
0.15
BOUND, (pmole/mg protein)
0.00
0.50
1.00
1.50
2.00
BOUND, (pmola/mg protein)
FIG. 7. Scatchard analysis of [125I]rhIGF-I (A) and [126I]rhIGF-II (B) binding to mammary microsomes from nonlactating (O) and lactating (A) cows. Each point represents the mean ± SEM for five cows. A, 667 jug/ml microsomal protein from each cow were incubated with 0.2 ng/ ml [126I]rhIGF-I and 0-33 ng/ml rhIGF-I for 48 h at 4 C in a total volume of 0.75 ml. B, 133 tig/va\ the microsomal protein from each cow were incubated with 0.2 ng/ml [125I]rhIGF-II and 0-33 ng/ml rhIGF-II for 48 h at 4 C in a total volume of 0.75 ml. Nonspecific binding was measured in the presence of a 1700-fold molar excess of unlabeled IGF. B/F, Bound to free ratio. TABLE 1. Affinity constants (Ka) and binding capacities (Bmax) for the IGF receptors in mammary microsomes isolated from prepartum and postpartum dairy cows
IGF-I Prepartum Postpartum IGF-II Prepartum Postpartum
Kd
B m ax
(nmol/liter)
(pmol/mg protein)
0.68 ± 0.08 0.71 ± 0.17
0.10 ± 0.01 0.16 ± 0.04
0.44 ± 0.03 0.47 ± 0.09
1.00 ± 0.17 1.68 ± 0.48
Values are the mean ± SEM (n = 5).
postpartum cows. However, visual analysis of the Scatchard plots leads us to suggest that any changes in specific binding that were observed in the survey binding data (Fig. 6) were probably due to changes in receptor number and not affinity. Furthermore, these data show that the binding capacity of the type II receptor is at least 10-fold greater than that of the type I receptor. Discussion High affinity receptors for IGFs occur in the bovine mammary gland. Affinity cross-linking studies with IGFI in bovine mammary microsomes have previously demonstrated that IGF-I binds to proteins of approximately 260 and 135 mol wt (5). These subunits have also been confirmed to bind IGF-I in the rabbit mammary gland,
641
while IGF-II binds only to a 220 mol wt subunit (1). Binding of IGF-I as a function of microsomal protein concentration followed the expected linear relationship with protein mass. However, the curvilinear relationship between bound IGF-II and microsomal protein concentration was unexpected, and was probably due to a much higher type II receptor concentration present over the range of microsomal protein used in these studies. The ability of IGF-I, IGF-II, and insulin to compete for type I and II receptors agrees with previous data collected from bovine (2), rabbit (1), and ewe (6) mammary tissue. Because recombinant IGF-I was used in these studies, cross-reactivity of rhIGF-I for the type II receptor was not observed. This finding agrees with previous work in bovine mammary tissue with rhIGF-I (2). Kinetic studies supported the conclusion that the binding reactions for both IGF-I and IGF-II are reversible, yet the dissociation reactions were about 4- to 5-fold slower than the association reactions. Scatchard analysis of the receptors in mammary microsomes showed the prevalence of type II receptor to be at least 10-fold higher than that of the type I receptor. This suggests greater sensitivity to IGF-II than IGF-I and agrees with the work done on mammary tissue from the cow (5) ewe (6), and rat (2). The decline in IGF-I binding that occurs prepartum may be due to one of two factors. Receptor down-regulation could cause the decline in type I receptors (1416). High concentrations of IGF-I have been found in the prepartum secretions of cows (17). Furthermore, IGF-I binding is known to induce type I receptor downregulation in several cell types (14-16). Presumably, the same principle is applicable to the cells within the mammary gland. Another possibility is that the population of cells changes within the mammary gland, altering the contribution of membrane to the microsomal pool by specific cell types. Mammary epithelial cell number declines during involution of the mammary gland and then increases as the animal approaches the onset of the next lactation (18). The increase in type I receptor activity with the onset of lactation may play a role in increasing the metabolic activity of the gland. This increase in type I receptors has also been observed through affinity cross-linking studies in bovine mammary microsomes (5). The Scatchard analysis conducted in this study suggests that the increase in IGF-I binding is due to increased receptor numbers. IGF-I is reported to stimulate responses in mammary tissue, such as increased synthesis of milk proteins, increased glucose transport (19), and increased DNA and lactose synthesis (20). Additionally, the increase in type I receptors would explain the report that
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IGF RECEPTORS IN MAMMARY TISSUE
lactating bovine mammary tissue synthesizes more DNA in vitro in response to insulin or IGF-I than nonlactating mammary tissue (Baumrucker, C. R., and B. H. Stemberger, manuscript in preparation). The mechanism behind the increase in IGF-I receptors at parturition is unclear. The increase in mammary epithelial cell number during the 2 weeks before parturition may be responsible for increased IGF receptors. However, the periparturient period is also associated with a dramatic surge in plasma lactogenic hormone concentrations (21), and perhaps the components of this surge elicit this receptor response. Glucocorticoids have been reported to increase IGF-I binding in human flbroblasts (22). PRL increases the sensitivity of mouse mammary tissue to insulin (23). Conceivably, a periparturient surge of glucocorticoids could up-regulate type I receptors, either directly or through the action of PRL. The change in binding that occurs during lactation may be due to a decline in epithelial cell number. Cell numbers increase during early lactation and then decline in later lactation (21). However, the ability of this survey to detect the short term change in binding that would result from this increase during early lactation is limited due to a lack of frequently spaced observations. Expression of the IGF receptors in the bovine mammary gland appears to change in accordance with the various metabolic adaptations that occur in the cow during lactogenesis, lactation, and involution. The increased IGF receptor expression observed in the cow with the onset of lactation indicates that IGFs may be important regulators of milk synthesis by the mammary gland. Of particular interest is the increase in milk production in the cow in response to exogenous GH (24). This response is observed in the absence of GH receptors in the mammary gland. Furthermore, exogenous GH elevates blood IGF-I concentrations in the cow (25). The presence of type I IGF receptors on the bovine mammary gland coupled with the elevated blood IGF-I concentrations in response to exogenous GH support the hypothesis that IGF-I may mediate GH action at the level of the mammary gland. However, the fact that the expression of IGF receptors decreases with the onset of lactation in the ewe (6) and rat (2) illustrates the need for caution when making inferences about the biological role of IGFs in lactation physiology. Because the dairy cow has been subjected to years of intensive genetic selection for high milk yield, the possibility exists for differences in mammary metabolism between the dairy cow and other species. Further studies on the role of IGFs in mammary function are necessary.
Acknowledgments The authors thank C. Anne Gibson and Stan Wampler for their assistance.
Endo • 1990 Voll26«Nol
References 1. Barenton B, Guyda HJ, Goodyer CG, Polychronakos C, Posner BI 1987 Specificity of insulin-like growth factor binding to type-II IGF receptors in rabbit gland and hypophysectolized rat liver. Biochem Biophys Res Commun 149:555 2. Collier RI, Ganguli S, Menke PT, Buonomo FC, McGrath ME, Kotts CE, Kn.M GG. 1989 Changes in insulin and somatomedin receptors and uptake of insulin, IGF-I and IGF-II during mammary growth, lactogenesis and lactation. In: Heap RB, Prosser CG, Lamming GE (eds) Biotechnology in Growth Regulation. Butterworth, London, p 153 3. Gregor P, Burleigh BD, Presence of high affinity somatomedin/ insulin-like growth factor receptors in porcine mammary gland. 67th Annual Meeting of The Endocrine Society, Baltimore MD, 1985, p 223 (Abstract) 4. Pekonen F, Partanen S, Makinen T, Rutanen E 1988 Receptors for epidermal growth factor and insulin-like growth factor I and their relation to steroid receptors in human breast cancer. Cancer Res 48:1343 5. Dehoff MH, Elgin RG, Collier RI, Clemmons DR 1988 Both type I and II insulin-like growth factor receptor binding increase during lactogenesis in bovine mamary tissue. Endocrinology 122:2412 6. Disenhaus C, Belair L Djiane J. 1988. Characterization and physiological development of the receptors for the insulin-like growth factors I and II (IGFs) in the mammary gland of the ewe. Reprod Nutr Dev 28:241 7. Gertler A, Ashkenazi A, Mader Z 1984 Binding sites of human growth hormone and ovine and bovine prolactins in the mammary gland and liver of lactating dairy cows. Mol Cell Endocrinol 34:51 8. Baumrucker CR, Growth hormone does not directly affect bovine mammary tissue growth nor lactating acini milk production in culture 80th Annual Meeting of the American Dairy Science Association, Urbana IL, 1985, p 106 (Abstract) 9. Salmon WD, Daughaday WH 1957 A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825 10. Pocius PA, Dreels JM, Devery-Pocius JE, Baumrucker CR 1984 Techniques for preparation of bovine mammary smooth membranes. J Dairy Sci 67:2055 11. Oscar TP, Baumrucker CR, Etherton TD 1986 Insulin binding to bovine mammary membranes: comparison of microsomes versus smooth membranes. J Anim Sci 62:179 12. Etherton TD, Wiggens JP, Evock CM, Chung CS, Rebhun JF, Walton PE, Steele NC 1987 Stimulation of pig growth performance by porcine growth hormone: determination of the dose-response relationship. J Anim Sci 64:433 13. Neter J, Wasserman W, Kutner MH 1985 Applied Linear Statistical Models, ed 2. Irwin, Homewood, IL, pp 328, 517 14. Rosenfeld RG, Hintz RL 1980 Characterization of a specific receptor for somatomedin C (SM-C) on cultures human lymphocytes: evidence that SM-C modulates homologous receptor concentration. Endocrinology 107:1848 15. Rosenfeld RG, Dollar I-A 1982 Characterizationof the somatomedin- C/insulin-like growth factor I (SM-C/IGF-I) receptor on human fibroblast monolayers: regulation of receptor concentrations by SM-C/IGF-I and insulin. J Clin Endocrinol Metab 55:434 16. Geory ES, Rosenfeld RG, Hoffman AR 1989 Insulin-like growth factor-I is internalized after binding to the type I insulin-like growth factor receptor. Horm Metab Res 21:1 17. Vega JR, Gibson CA, Baumrucker CR, Insulin-like growth factor I (IGF-I) mammary secretions of prepartum dairy cows. 84th Annual Meeting of the American Dairy Science Association, Lexington KY, 1989, p 194 (Abstract) 18. Sordillo LM, Nickerson SC 1988 Morphologic changes in the bovine mammary gland during involution and lactogenesis. Am J Vet Res 49:1112 19. Prosser CG, Sankaran L, Hennighausen L, Topper YJ 1987 Comparison of the roles of insulin and insulin-like growth factor I in casein gene expression and in the development of alpha-lactalbumin and glucose transport activities in the mouse mammary epithelial cell. Endocrinology 120:1411
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IGF RECEPTORS IN MAMMARY TISSUE 20. Baumrucker CR, Insulin like growth factor I (IGF-I) and insulin stimulate lactating bovine mammary tissue DNA synthesis and milk production in vitro. 81st Annual Meeting of the American Dairy Science Association, Davis CA, 1986, p 120 (Abstract) 21. Tucker HA 1981 Physiological control of mammary growth, lactogenesis and lactation. J Dairy Sci 64:1403 22. Klapowitz PB 1987 Glucocorticoids enhance somatomedin-C binding and stimulation of amino acid uptake in human fibroblasts. J Clin Endocrinol Metab 64:563
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23. Oka T, Topper YJ 1972 Is prolactin mitogenic for mammary epithelium? Proc Natl Acad Sci USA 69:1693 24. Peel CJ, Bauman DE, Gorewit RC, Sniffen CI 1981 Effect of growth hormone on lactational performance in high yielding dairy cows. J Nutr 111:1662 25. Peel CJ, Sandles LD, Quelch KH, Herington AC 1985 The efiects of long term administration of bovine growth hormone on the lactational performance of identical twin dairy cows. Anim Prod 41:135
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Vol. 126, No. 1 Printed in U.S.A.
Characterization of the Change in Type I and II InsulinLike Growth Factor Receptors of Bovine Mammary Tissue during the Pre- and Postpartum Periods* DARRYL L. HADSELL, PHIL G. CAMPBELL, AND CRAIG R. BAUMRUCKER Department of Dairy and Animal Science, Pennsylvania State University (D.L.H., C.R.B.), University Park, Pennsylvania 16802; and the Allegheny-Singer Research Institute ((P.G.C.), Pittsburgh, Pennsylvania 15212-9986
ABSTRACT. Type I and II receptors for the insulin-like growth factors (IGF) were characterized in microsomes from bovine mammary tissue. Specific binding of [125I]IGF-I increased linearly with increasing microsomal protein concentrations. In contrast, IGF-II binding showed a curvilinear relationship over the concentration range tested, with a maximum at 120 ng/va\. Kinetic studies conducted at 4 C showed the binding reactions to be reversible. Competition studies showed recombinant human IGF-I (rh-IGF-I) to be 8-, 18-, and 1000-fold more potent at inhibiting [125I]rh-IGF-I binding than recombinant bovine IGF-II (rb-IGF-II), multiplication-stimulating activity, and insulin, respectively. rbIGF-II was 1.8- and 6-fold more potent at inhibiting [125I]rbIGF-II binding than rhIGF-II and multiplication-stimulating activity. Specific binding of [125I]IGF-I and -II
was measured in microsomes prepared from cows (n = 47) ranging from 138 days prepartum to 411 days postpartum. IGFI binding declined during the prepartum period, increased 75% with the onset of lactation, and then declined during the postpartum period. Scatchard analysis showed the presence of a single class of high affinity binding sites for both ligands, with type II receptors being about 10-fold more prevalent than type I receptors. The survey and Scatchard data support the conclusion that the onset of lactation coincides with an increase in the number of type I receptors present in mammary tissue. This increase supports the contention that IGF-I may play an important role in modulating the metabolic activity of the bovine mammary gland. {Endocrinology 126: 637-643, 1990)
R
(4). Further, lactogenesis in the cow is associated with increased binding of IGF-I to mammary microsomes (5). In contrast, type I receptors are higher during gestation for the rat and ewe than they are during lactation (2, 6). The type II receptors are higher in pregnant and lactating rats than in virgin rats, with no apparent change in type II receptors with the onset of lactation. In the ewe the type II receptors are lower during lactation than they are during the prepartum period. These findings, while varied across species, suggest that the IGF receptors play an important role in modulation of the growth and secretory function of the mammary gland. The objectives of this study were to characterize in greater detail the receptors for IGFs in the microsomal fraction of bovine mammary tissue. The focus of these studies was on specificity, kinetic properties, affinity, and binding capacity of the IGF receptors in the mammary gland during the prepartum (nonlactating) and postpartum (lactating) periods.
ECEPTORS for insulin-like growth factors (IGFs) have been demonstrated in mammary tissue from rabbit (1), rat (2), pig (3), human (4), cow (5), and ewe (6). In the cow these receptors are currently believed to be involved in the IGF-mediated actions of GH on the mammary gland (5). This belief is based on the failure to detect receptors for GH in bovine mammary tissue (7), and the failure of GH to stimulate growth of or milk secretion by bovine mammary tissue in vitro (8). The mechanism by which IGFs could mediate the GH response in mammary tissue is analogous to a hypothesis that was initially proposed to describe stimulation of bone growth by GH (9). Mammary gland IGF receptors have been reported to change with the physiological state of the tissue. For example, transformation of human mammary tissue to a malignant state coincides with increased IGF-I binding Received August 21, 1989. Address requests for reprints to: Dr. Craig R. Baumrucker, Department of Dairy and Aninial Science, Pennsylvania State University, 302 Henning Building, University Park, Pennsylvania 16802. * This work was supported by the Pennsylvania Agricultural Experimental Station and USDA Grant 85-CRCR-1-1881. Authorization for publication as paper 8278 in the journal series of the Pennsylvania Agricultural Experimental Station.
Materials and Methods Materials Recombinant bovine IGF-II (rb-IGF-II) was a generous gift of Dr. Robert Collier (Monsanto Corp., St. Louis, MO). Recom637
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IGF RECEPTORS IN MAMMARY TISSUE
638
binant human IGF-I (rh-IGF-I) was a generous gift of Dr. Dan Burleigh (Pitman-Moore, Inc., Terre Haute, IN). rh-IGF-II was purchased from Bachem, Inc. (Torrence, CA). Na125I was obtained from Amersham International (Arlington Heights, IL). Porcine proinsulin was obtained from Eli Lily Co. (Indianapolis, IN). Bovine GH (bGH) and bovine PRL (bPRL) were gifts from the NIDKK. All other reagents were of analytical grade. Preparation of microsomes Mammary tissue was collected from prepartum and postpartum dairy cows that were slaughtered by the Pennsylvania State University Dairy Research Facility for reasons other than mastitis. All cows in this study had one or more lactations. The tissue was placed on ice for transport from the slaughter facility to the laboratory and then frozen at -20 C until preparation of microsomes. Microsomes were prepared as described by Pocius et al. (10) and then stored at -80 C. The protein concentration of the microsome preparations was determined by the bicinchoninic acid assay (Pierce, Rockford, IL). IGFRRA IGF receptor-binding activity was measured using a modification of the insulin RRA of Oscar et al. (11). IGF-I and -II were iodinated to a specific activity of 100-300 ^Ci/jug, using chloramine-T and purified by the method of Etherton et al. (12). The assay was performed using 12 X 75-mm polypropylene tubes in 750 n\ 50 mM Tris butfer (pH 7.8) containing 0.5% BSA. Microsomal protein was incubated with 0.2 ng/ml [125I] IGF-I or -II for 48 h at 4 C unless otherwise stated. Receptorbound hormone was separated from free by centrifugation at 3000 x g at 4 C for 30 min. The pellet was counted in a model 1274 RIAGAMMA counter (LKB Instruments, Rockville, MD). Nonspecific binding was determined in the presence of 333 ng/ml unlabeled IGF-I or -II.
Endo • 1990 Voll26«Nol
Data analysis Scatchard plots for IGF-I and -II binding were fit by least squares linear regression. The kinetic and inhibition data were analyzed with the Graphpad data analysis software package (ISI Software, Philadelphia, PA). Analysis of the survey data was performed by discontinuous least squares linear regression. The Scatchard parameter estimates from pre- and postpartum cows were analyzed by a one-way analysis of variance (13).
Results Protein dependence Both total and specific binding of [125I] IGF-I (Fig. 1A) increased in a linear fashion over the range of microsomal protein tested. From this study 667 /u.g/ml microsomal protein were selected for all subsequent IGF-I RRAs. At this concentration of microsomal protein nonspecific binding was about 30% of total binding. Total binding of [125I]IGF-II as a function of microsomal protein concentration (Fig. IB) increased in a hyperbolic fashion (y = 0.27[1 - e0017x] + 0.148; P < 0.005), approaching an asymptotic maximum of 41% of the total counts added at 667 fig/ml membrane protein. Nonspecific binding increased in a linear fashion (r2 = 0.92). Specific binding as a function of microsomal protein was best fit by a mixed exponential function (y = 0.07[e-°007x] + 0.26[l - e"003*] - 0.0004; P < 0.005), showing a maximum at 120 ng/m\. At this dose of micro0.15-,
A
0.12 0.09
Receptor characterization
0.06
The protein dependence of [125I]IGF binding was assayed by testing 133-880 Mg/ml microsomal protein for rhIGF-I and 9667 Mg/ml for rbIGF-II. The time dependence of association was determined at 4 C with 667 Mg/ml microsomal protein for rhIGF-I and 133 Mg/ml microsomal protein for rbIGF-II. The dissociation reaction was followed for 96 h after adding a 1700fold molar excess IGF-I or -II to microsomes that were preequilibrated with [125I]IGF-I or -II for 48 h. The specificity of [125I]rhIGF-I and [125I]rbIGF-II binding was measured in the presence of varying concentrations of rhIGF-I, rbIGF-II, rhIGF-II, bovine insulin, multiplication-stimulating activity (MSA), porcine proinsulin, bGH, and bPRL. A survey of specific binding of [125I]rhIGF-I or [125I]rbIGF-II was carried out with microsomes from 46 individual cows ranging from -138 to 411 days relative to parturition. The concentration of microsomal protein for both IGF-I and -II was 667 Mg/ml. Scatchard analysis was performed on the binding of rhIGF-I and rhlGFII from 5 prepartum and 5 postpartum cows using 667 Mg/ml microsomal protein for IGF-I and 133 Mg/ml microsomal protein for IGF-II. The concentration of unlabeled IGF-I or -II in both assays ranged from 0-33 ng/ml.
0.03 ^ 0.00 200
400
600
800
MICROSOMAL PROTEIN. (jiq/m\)
0.40 0.300.20 0.10
0.00 100
200 300 400 500 600 700 MICROSOMAL PROTEIN, (jtq/m\)
800
FIG. 1. Total (O), specific (0), and nonspecific (A) binding of [125I] rhIGF-I (A) and [125I]rbIGF-II (B) as a function of mammary microsome concentration. Each point represents the mean ± SEM for three cows. Various concentrations of microsomal protein were incubated with 0.2 ng/ml [125I]rhIGF-I or [125I]rbIGF-II in a total volume of 0.75 ml for 48 h at 4 C. Nonspecific binding was measured in the presence of a 1700-fold molar excess of IGF-I or -II. B/T, Bound to total ratio.
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IGF RECEPTORS IN MAMMARY TISSUE somal protein, the nonspecific binding of IGF-II was about 25% of the total binding. Unless otherwise stated, 133 Mg/ml microsomal protein was chosen for all IGF-II RRAs.
639
g 60.0a. l" 40.0E
Association/dissociation [125I]IGF-I associated (Fig. 2A) to the membranes with a rate constant of 0.26 fmol/mg protein-h, approaching an asymptotic maximum of 1.86 fmol/mg protein. Equilibrium was reached for this reaction within 24 h. The rate of [125I]IGF-I dissociation (Fig. 2B) was 0.06 fmol/ mg protein-h. About 30% of the [125I]IGF-I bound at equilibrium remained bound 96 h after the addition of excess unlabeled IGF-I. [125I]IGF-II binding increased (Fig. 3A) with a rate constant of 0.14 fmol/mg proteinh and reached an asymptotic maximum of 44.61 fmol/ mg protein. Equilibrium was reached by about 30 h. The dissociation of [125I]IGF-II (Fig. 3B) proceeded with a rate constant of 0.03 fmol/mg protein-h. About 42% of 3.01
J I
FiG. 3. Association (A) and dissociation (B) kinetics for the binding of [125I]rbIGF-II to mammary microsomes. Each point represents the mean ± SEM for two cows. A, 133 Mg/ml microsomal protein were incubated with 0.2 ng/ml [125I]rbIGF-II for various times at 4 C in a total volume of 0.75 ml. B, 133 Mg/ml microsomal protein were equilibrated for 48 h with 0.2 ng/ml [125I]rbIGF-II in a total volume of 0.75 ml. After the equilibration step, a 1700-fold molar excess of rblGF-II was added to each tube, and binding was measured at various times.
the [125I] IGF-II bound at equilibrium remained bound to the membranes by 96 h.
I I
20
TIME, (h)
Specificity
60
40
80
TIME, (h) 2.0 n
40
60
100
TIME, (h)
FIG. 2. Association (A) and dissociation (B) kinetics for the binding of [126I]rhIGF-I to mammary microsomes. Each point represents the mean ± SEM for two cows. A, 667 Mg/ml microsomal protein in a total volume of 0.75 ml were incubated with 0.2 ng/ml [125I]rhIGF-I for various times at 4 C. B, 667 Mg/ml microsomal protein were equilibrated for 48 h with 0.2 ng/ml [125I]rhIGF-I in a total volume of 0.75 ml. After the equilibration step, a 1700-fold molar excess of rhIGF-I was added to each tube, and binding was measured at various times.
Specific binding of [125I]rhIGF-I (Fig. 4A) in the presence of 0-667 ng/ml rhIGF-I, rblGF-II, or MSA showed ED50 values of 3.6 ± 1.4, 30.1 ± 1.5, and 67.3 ± 15 ng/ ml, respectively. The ED50 of insulin for inhibition of [125I]rhIGF-I binding was 3.6 ± 0.002 Mg/ml. Proinsulin could only inhibit about 25% of [125I]rhIGF-I binding at a concentration of 6.7 jug/ml. Neither bGH nor bPRL could inhibit [125I]rhIGF-I binding at concentrations of up to 13 Mg/ml (data not shown). Specific binding of [125I]rbIGF-II (Fig. 4B) was measured with 667 fig/ml microsomal protein in the presence of 0-133 ng/ml rblGF-II or 0-267 ng/ml MSA. The ED50 values for rblGF-II and MSA were 13.6 ± 1.4 and 82.34 ± 1 . 1 ng/ml, respectively. rhIGF-I, insulin, proinsulin, GH, and PRL failed to inhibit [125I]rbIGF-II binding at concentrations of up to 0.67, 67, 6.60, 13, and 13 ng/ml, respectively (data shown for rhIGF-I only). In a separate IGF-II RRA, the potency of rhIGF-II was compared with that of rblGF-II using 133 Mg/ml microsomal protein and [125I]rbIGF-II The ED50 for rblGF-II in this case was 13.2 ± 1.1 ng/ml, while that for rhIGF-II was 25.1 ± 1.0 ng/ml (Fig. 5). Pre- and postpartum IGF-binding activity Binding of [125I]IGF-I and -II as a function of physiological state and days relative to parturition is presented
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IGF RECEPTORS IN MAMMARY TISSUE
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Endo • 1990 Voll26«Nol
1.25T
1.00 0.75 0.50 0.25
1.0E-2
0.1
1.0
10.0
100.0 1000.0 1.0E4
1.0E5
COMPETITOR, (ng/ml)
B
1.00
*> -c^uur p " ° ° o o o £^JTUF o
0.75-
-150-100 -50
0
0.50-
\
50
100 150 200 250 300 350 400 450
TIME, (days postpartum)
\
0.25
\ 0.00
1.0E-2
0.1
^
100-,
1.0 10.0 100.0 1000.0 1.0E4 COMPETITOR, (ng/ml)
1.0E5
FIG. 4. Inhibition of [125I]rhIGF-I (A) and [12SI]rbIGF-II (B) binding to pooled mammary microsomes (five nonlactating and five lactating cows) by rhIGF-I (O), rbIGF-II (A), MSA (•), bovine insulin (V), and porcine proinsulin (0). A, 667 fig/ml microsomal protein were incubated with 0.2 ng/ml [125I]rhIGF-I and various concentrations of the above competitors (see Materials and Methods) for 48 h at 4 C in a total volume of 0.75 ml. B, 667 jig/ml microsomal protein were incubated with 0.2 ng/ml [125I]rbIGF-II and various concentrations of competitors for 48 h at 4 C in a total volume of 0.75 ml. B/BO, Ratio of bound in the presence of competitor to bound in the absence of competitor.
«
80
e £ 60\ "5 O .E 40
O.
oo
20-
-150-100 -50 1.25y
o o
O
0
50
100 150 200 250 300 350 400 450
TIME, (days postpartum)
FIG. 6. Specific binding of [125I]rhIGF-I (A) and [126I]rbIGF-II (B) to mammary microsomes as a function of physiological state (nonlactating vs. lactating) and days relative to parturition. Each point represents the binding to a microsome preparation from one cow. Binding was measured after incubation of 667 Mg/ml microsomal protein with 0.2 ng/ml [125I]rhIGF-I or [125I]rbIGF-II for 48 h at 4 C in a total volume of 0.75 ml.
1.00-
0.75-
0.50-
1
10
100
1000
COMPETITOR, (ng/ml)
125
FIG. 5. Inhibition of [ I]rbIGF-I binding to pooled mammary nucrosomes (five nonlactating and five lactating cows) by rblGF-II-(A) and rhIGF-II (0). A 48-h incubation of 0.2 ng/ml [125I]rbIGF-II with 133 jig/ml microsomal protein and various concentrations of rbIGF-II or rhIGF-II (see Materials and Methods) was conducted at 4 C. B/BO, Bound to free ratio.
in Fig. 6. The data were analyzed by least squares linear regression. Regression of bound [125I]IGF-I on days relative to partuntion resulted in a significant fit of the model (P < 0.01). Both the intercept and the slope of the regression line for the postpartum group were different from those for the prepartum group. These data show that IGF-I binding decreased during the prepartum period, increased with the onset of lactation, and then decreased during the postpartum period.. IGF-I binding in the prepartum group declined at a rate of 0.025 fmol/ mg protein • day and intersected the y-axis at 3.07 fmol/ mg protein-day. Binding in the postpartum group declined by 0.004 fmol/mg protein • day and intersected the
y-axis at 5.4 fmol/mg protein. [125I]IGF-II binding appeared to follow the same pattern as IGF-I binding. However, due to larger cow to cow variation and an apparently smaller difference between the pre- and postpartum groups, no change in IGF-II binding was detected with the onset of lactation or with time during the preor postpartum period (Fig. 6B). Scatchard analysis of pre- and postpartum binding A comparison of the estimated Kd and binding capacity for [125I]IGF binding was obtained by Scatchard analysis of the binding data from five prepartum (-40 ± 18 days) and five postpartum (21.6 ± 12.4 days) cows (Fig. 7). Both receptors behaved as a single class of high affinity sites. The affinity and binding capacity of the IGF receptors are presented in Table 1. The analysis of variance failed to detect a significant difference in affinity or binding capacity for either ligand between the pre- and
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IGF RECEPTORS IN MAMMARY TISSUE 0.12 0.10 0.08 0.060.04 0.02 0.00 0.05
0.10
0.15
BOUND, (pmole/mg protein)
0.00
0.50
1.00
1.50
2.00
BOUND, (pmola/mg protein)
FIG. 7. Scatchard analysis of [125I]rhIGF-I (A) and [126I]rhIGF-II (B) binding to mammary microsomes from nonlactating (O) and lactating (A) cows. Each point represents the mean ± SEM for five cows. A, 667 jug/ml microsomal protein from each cow were incubated with 0.2 ng/ ml [126I]rhIGF-I and 0-33 ng/ml rhIGF-I for 48 h at 4 C in a total volume of 0.75 ml. B, 133 tig/va\ the microsomal protein from each cow were incubated with 0.2 ng/ml [125I]rhIGF-II and 0-33 ng/ml rhIGF-II for 48 h at 4 C in a total volume of 0.75 ml. Nonspecific binding was measured in the presence of a 1700-fold molar excess of unlabeled IGF. B/F, Bound to free ratio. TABLE 1. Affinity constants (Ka) and binding capacities (Bmax) for the IGF receptors in mammary microsomes isolated from prepartum and postpartum dairy cows
IGF-I Prepartum Postpartum IGF-II Prepartum Postpartum
Kd
B m ax
(nmol/liter)
(pmol/mg protein)
0.68 ± 0.08 0.71 ± 0.17
0.10 ± 0.01 0.16 ± 0.04
0.44 ± 0.03 0.47 ± 0.09
1.00 ± 0.17 1.68 ± 0.48
Values are the mean ± SEM (n = 5).
postpartum cows. However, visual analysis of the Scatchard plots leads us to suggest that any changes in specific binding that were observed in the survey binding data (Fig. 6) were probably due to changes in receptor number and not affinity. Furthermore, these data show that the binding capacity of the type II receptor is at least 10-fold greater than that of the type I receptor. Discussion High affinity receptors for IGFs occur in the bovine mammary gland. Affinity cross-linking studies with IGFI in bovine mammary microsomes have previously demonstrated that IGF-I binds to proteins of approximately 260 and 135 mol wt (5). These subunits have also been confirmed to bind IGF-I in the rabbit mammary gland,
641
while IGF-II binds only to a 220 mol wt subunit (1). Binding of IGF-I as a function of microsomal protein concentration followed the expected linear relationship with protein mass. However, the curvilinear relationship between bound IGF-II and microsomal protein concentration was unexpected, and was probably due to a much higher type II receptor concentration present over the range of microsomal protein used in these studies. The ability of IGF-I, IGF-II, and insulin to compete for type I and II receptors agrees with previous data collected from bovine (2), rabbit (1), and ewe (6) mammary tissue. Because recombinant IGF-I was used in these studies, cross-reactivity of rhIGF-I for the type II receptor was not observed. This finding agrees with previous work in bovine mammary tissue with rhIGF-I (2). Kinetic studies supported the conclusion that the binding reactions for both IGF-I and IGF-II are reversible, yet the dissociation reactions were about 4- to 5-fold slower than the association reactions. Scatchard analysis of the receptors in mammary microsomes showed the prevalence of type II receptor to be at least 10-fold higher than that of the type I receptor. This suggests greater sensitivity to IGF-II than IGF-I and agrees with the work done on mammary tissue from the cow (5) ewe (6), and rat (2). The decline in IGF-I binding that occurs prepartum may be due to one of two factors. Receptor down-regulation could cause the decline in type I receptors (1416). High concentrations of IGF-I have been found in the prepartum secretions of cows (17). Furthermore, IGF-I binding is known to induce type I receptor downregulation in several cell types (14-16). Presumably, the same principle is applicable to the cells within the mammary gland. Another possibility is that the population of cells changes within the mammary gland, altering the contribution of membrane to the microsomal pool by specific cell types. Mammary epithelial cell number declines during involution of the mammary gland and then increases as the animal approaches the onset of the next lactation (18). The increase in type I receptor activity with the onset of lactation may play a role in increasing the metabolic activity of the gland. This increase in type I receptors has also been observed through affinity cross-linking studies in bovine mammary microsomes (5). The Scatchard analysis conducted in this study suggests that the increase in IGF-I binding is due to increased receptor numbers. IGF-I is reported to stimulate responses in mammary tissue, such as increased synthesis of milk proteins, increased glucose transport (19), and increased DNA and lactose synthesis (20). Additionally, the increase in type I receptors would explain the report that
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IGF RECEPTORS IN MAMMARY TISSUE
lactating bovine mammary tissue synthesizes more DNA in vitro in response to insulin or IGF-I than nonlactating mammary tissue (Baumrucker, C. R., and B. H. Stemberger, manuscript in preparation). The mechanism behind the increase in IGF-I receptors at parturition is unclear. The increase in mammary epithelial cell number during the 2 weeks before parturition may be responsible for increased IGF receptors. However, the periparturient period is also associated with a dramatic surge in plasma lactogenic hormone concentrations (21), and perhaps the components of this surge elicit this receptor response. Glucocorticoids have been reported to increase IGF-I binding in human flbroblasts (22). PRL increases the sensitivity of mouse mammary tissue to insulin (23). Conceivably, a periparturient surge of glucocorticoids could up-regulate type I receptors, either directly or through the action of PRL. The change in binding that occurs during lactation may be due to a decline in epithelial cell number. Cell numbers increase during early lactation and then decline in later lactation (21). However, the ability of this survey to detect the short term change in binding that would result from this increase during early lactation is limited due to a lack of frequently spaced observations. Expression of the IGF receptors in the bovine mammary gland appears to change in accordance with the various metabolic adaptations that occur in the cow during lactogenesis, lactation, and involution. The increased IGF receptor expression observed in the cow with the onset of lactation indicates that IGFs may be important regulators of milk synthesis by the mammary gland. Of particular interest is the increase in milk production in the cow in response to exogenous GH (24). This response is observed in the absence of GH receptors in the mammary gland. Furthermore, exogenous GH elevates blood IGF-I concentrations in the cow (25). The presence of type I IGF receptors on the bovine mammary gland coupled with the elevated blood IGF-I concentrations in response to exogenous GH support the hypothesis that IGF-I may mediate GH action at the level of the mammary gland. However, the fact that the expression of IGF receptors decreases with the onset of lactation in the ewe (6) and rat (2) illustrates the need for caution when making inferences about the biological role of IGFs in lactation physiology. Because the dairy cow has been subjected to years of intensive genetic selection for high milk yield, the possibility exists for differences in mammary metabolism between the dairy cow and other species. Further studies on the role of IGFs in mammary function are necessary.
Acknowledgments The authors thank C. Anne Gibson and Stan Wampler for their assistance.
Endo • 1990 Voll26«Nol
References 1. Barenton B, Guyda HJ, Goodyer CG, Polychronakos C, Posner BI 1987 Specificity of insulin-like growth factor binding to type-II IGF receptors in rabbit gland and hypophysectolized rat liver. Biochem Biophys Res Commun 149:555 2. Collier RI, Ganguli S, Menke PT, Buonomo FC, McGrath ME, Kotts CE, Kn.M GG. 1989 Changes in insulin and somatomedin receptors and uptake of insulin, IGF-I and IGF-II during mammary growth, lactogenesis and lactation. In: Heap RB, Prosser CG, Lamming GE (eds) Biotechnology in Growth Regulation. Butterworth, London, p 153 3. Gregor P, Burleigh BD, Presence of high affinity somatomedin/ insulin-like growth factor receptors in porcine mammary gland. 67th Annual Meeting of The Endocrine Society, Baltimore MD, 1985, p 223 (Abstract) 4. Pekonen F, Partanen S, Makinen T, Rutanen E 1988 Receptors for epidermal growth factor and insulin-like growth factor I and their relation to steroid receptors in human breast cancer. Cancer Res 48:1343 5. Dehoff MH, Elgin RG, Collier RI, Clemmons DR 1988 Both type I and II insulin-like growth factor receptor binding increase during lactogenesis in bovine mamary tissue. Endocrinology 122:2412 6. Disenhaus C, Belair L Djiane J. 1988. Characterization and physiological development of the receptors for the insulin-like growth factors I and II (IGFs) in the mammary gland of the ewe. Reprod Nutr Dev 28:241 7. Gertler A, Ashkenazi A, Mader Z 1984 Binding sites of human growth hormone and ovine and bovine prolactins in the mammary gland and liver of lactating dairy cows. Mol Cell Endocrinol 34:51 8. Baumrucker CR, Growth hormone does not directly affect bovine mammary tissue growth nor lactating acini milk production in culture 80th Annual Meeting of the American Dairy Science Association, Urbana IL, 1985, p 106 (Abstract) 9. Salmon WD, Daughaday WH 1957 A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825 10. Pocius PA, Dreels JM, Devery-Pocius JE, Baumrucker CR 1984 Techniques for preparation of bovine mammary smooth membranes. J Dairy Sci 67:2055 11. Oscar TP, Baumrucker CR, Etherton TD 1986 Insulin binding to bovine mammary membranes: comparison of microsomes versus smooth membranes. J Anim Sci 62:179 12. Etherton TD, Wiggens JP, Evock CM, Chung CS, Rebhun JF, Walton PE, Steele NC 1987 Stimulation of pig growth performance by porcine growth hormone: determination of the dose-response relationship. J Anim Sci 64:433 13. Neter J, Wasserman W, Kutner MH 1985 Applied Linear Statistical Models, ed 2. Irwin, Homewood, IL, pp 328, 517 14. Rosenfeld RG, Hintz RL 1980 Characterization of a specific receptor for somatomedin C (SM-C) on cultures human lymphocytes: evidence that SM-C modulates homologous receptor concentration. Endocrinology 107:1848 15. Rosenfeld RG, Dollar I-A 1982 Characterizationof the somatomedin- C/insulin-like growth factor I (SM-C/IGF-I) receptor on human fibroblast monolayers: regulation of receptor concentrations by SM-C/IGF-I and insulin. J Clin Endocrinol Metab 55:434 16. Geory ES, Rosenfeld RG, Hoffman AR 1989 Insulin-like growth factor-I is internalized after binding to the type I insulin-like growth factor receptor. Horm Metab Res 21:1 17. Vega JR, Gibson CA, Baumrucker CR, Insulin-like growth factor I (IGF-I) mammary secretions of prepartum dairy cows. 84th Annual Meeting of the American Dairy Science Association, Lexington KY, 1989, p 194 (Abstract) 18. Sordillo LM, Nickerson SC 1988 Morphologic changes in the bovine mammary gland during involution and lactogenesis. Am J Vet Res 49:1112 19. Prosser CG, Sankaran L, Hennighausen L, Topper YJ 1987 Comparison of the roles of insulin and insulin-like growth factor I in casein gene expression and in the development of alpha-lactalbumin and glucose transport activities in the mouse mammary epithelial cell. Endocrinology 120:1411
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