We have proposed that growth hormone (GH) and prolactin (PRL) interact to suppress apoptosis in the mammary gland. GH increases insulin-like growth factor-I (IGF-I) synthesis whereas PRL suppresses the production of insulin-like growth factor-binding protein-5 (IGFBP-5) in the epithelial cells, which would otherwise inhibit IGFmediated cell survival. IGFBP-5 was present in milk from involuting glands at high concentrations (approximately 60 µg/ml) and had a high affinity (8·03 10 10 M) for IGF-I, suggesting an inhibitory effect of IGFBP-5 in the mammary gland. IGFBP-5 was present in the micellar fraction of milk and binds specifically to s2 -casein. Since s2 -casein also binds plasminogen and tissue-type plasminogen activator (t-PA), resulting in the conversion of plasminogen to plasmin, and since IGFBP-5 binds to plasminogen activator inhibitor-1 (PAI-1), we investigated whether apoptosis and extracellular matrix (ECM) degradation might be coordinately controlled by GH and PRL possibly acting through IGFBP-5.Litters were removed from lactating rats to initiate involution. Plasminogen activation and t-PA activity were both increased dramatically after 48 h and GH and PRL suppressed this response. By contrast, 17 -oestradiol, progesterone or corticosterone did not influence either process. An antiserum to IGF-I, which blocked systemic IGF-I effects, failed to inhibit the activation of plasminogen or the increase in t-PA, suggesting that paracrine effects of IGF-I may be more important. Teat-sealing, which led to the accumulation of milk without hormonal changes, also led to increases in plasminogen activation and t-PA activity, suggesting that locally produced factors (of which IGFBP-5 is one) are important in controlling ECM remodelling. We propose that GH and PRL inhibit apoptosis and ECM remodelling by a process that involves the control of IGF-I and PAI-1 availability by IGFBP-5, thus allowing these processes to be tightly coordinated.

Both PRL and GH play a role in maintaining lactation in the rat, although GH can only maintain pup weight gain at around 50% of the control value, whereas PRL can maintain weight gain close to 90% in the absence of GH. In this study we examined the effects of PRL and GH deficiency (using bromocriptine and an antiserum to rat GH) on milk yield and composition in lactating rats. Treatment with bromocriptine to suppress PRL secretion for 48 h led to a 57% decrease in milk yield with a concomitant decrease in milk protein and lactose yields, but no decrease in fat output. This led to the production of milk with a lower lactose concentration but increased concentrations of protein and particularly fat (increased 100%), which suggests that GH serves an auxiliary role by maintaining an energy-rich milk for the neonate when PRL secretion is reduced. This decrease in milk synthesis was accompanied by decreases in total mammary DNA content and increased milk sodium concentrations. The latter indicates the opening of tight junctions between mammary epithelial cells, which normally occurs during dedifferentiation and involution of the mammary gland. This suggests that PRL maintains milk synthesis at least in part by inhibiting epithelial cell loss and maintaining cellular differentiation. A deficiency in GH, by contrast, caused only a small decrease (24%) in milk yield and had no effect on the major constituents of milk or on milk sodium concentrations or total mammary DNA content. When animals were made deficient in both PRL and GH, however, there was a further marked decrease (88%) in milk volume along with the yields of all major milk constituents, confirming our previous findings that PRL and GH are the major regulators of milk synthesis. Recent studies have indicated that GH exerts direct effects on mammary gland growth, but its actions on milk secretion have been proposed to be mediated indirectly via insulin-like growth factor-I (IGF-I). We, therefore, inhibited lactation by inducing PRL and GH deficiency for 48 h and then attempted to reinitiate it by administering GH either systemically or by local oil-based implants into the mammary gland. Oil-based GH implants were as effective in stimulating milk secretion in the treated (but not contralateral, control) gland as was systemic GH treatment. Thus, GH does act directly on the mammary gland to stimulate milk synthesis, although this does not rule out the possibility that GH acts by stimulating local production of IGF-I.(ABSTRACT TRUNCATED AT 400 WORDS)

The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in IGF binding, but only recently have some of the critical residues in the IGFBP sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat IGFBP-5 that are involved in binding IGF-I. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of rIGFBP-5. Relative to wild-type rIGFBP-5, an 8-fold reduction in affinity for human IGF-I was found for recombinant G203K protein in both IGF-I ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in IGFBP-5 are important determinants in binding IGF-I, and due to their complete conservation in all IGFBP sequences, we suggest that they are likely to be involved in binding IGF-I in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of IGFBP-5, demonstrating that the C-terminus contains partially overlapping IGF-I and ECM binding sites. We therefore propose that heparin binding to basic amino acids in IGFBP-5 between 201-218 may physically occlude subsequent interaction between IGF-I and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound IGFBP-5 for IGF-I.

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Young growing rats treated with an antiserum to adipocytes showed marked reductions in several adipose tissue depots but surprisingly demonstrated increased body weight gain. During the first 3 wk after treatment body weight gain increased by 17% with no effect on food intake, whereas during weeks 3-7 body weight gain increased by 40-50% and was accompanied by a 15% increase in food intake. These animals thus exhibited increased food conversion efficiencies (intake/gain) of approximately 15-20% for almost 2 mo. Subsequently, food intake and body weight gain returned to normal (although body weight remained elevated) up to 6 mo. By this time several fat depots were still reduced in size, although total (chemical) fat was identical in treated and control groups. These results suggest that 1) reduction of body fat depots can be achieved using antibodies to adipocytes, 2) compensatory increases in lean body mass can occur, and 3) total fat mass may be regulated.