Characterization of a Somatomedin (Insulin-like Growth Factor) Synthesized by Fetal Rat Liver Organ Cultures*

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.


Explants
of 19-to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulinlike growth factor) BRL-MSA, multiplication-stimulating activity.
Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.
Growth hormone, the primary determinant of linear skeletal growth in childhood, is thought to act on cartilage via mediating polypeptides known as somatomedins or insulin-like growth factors (1). Four somatomedins have been purified from human plasma: IGF-I (2,3),' IGF-II (2) (4), and somatomedin C (5). A fifth member of this family of polypeptides, multiplication-stimulating activity (MSA), has been purified to homogeneity from serum-free media conditioned by the BRL 3A rat liver cell line (6-9). The properties of the somatomedins include: 1) single chain, acid-soluble polypeptides of molecular weight 5,000 to 10,000; 2) weak insulin-like activity in adipose tissue in vitro; 3) stimulation of DNA synthesis in and multiplication of fibroblasts in culture; 4) stimulation of sulfate uptake by cartilage in uitro; 5) interaction with cell surface receptors specific for somatomedins and weak interaction with insulin receptors; 6) binding to specific somatomedin carrier proteins in rat and human serum; 7) cross-reactivity in specific immunoassays for somatomedins (10).
By contrast, growth hormone does not appear to be an important determinant of fetal growth. Maternal growth hormone is present at low levels and does not cross the placental barrier, and fetuses with growth hormone deficiency are born normal in size (11). A possible role for somatomedins as fetal growth substances has been suggested recently. Somatomedins are present in cord blood and in amniotic fluid (12)(13)(14)(15)(16)(17)(18)(19)(20), and fetal tissues contain specific receptors for somatomedins (14). To help elucidate the role of somatomedins in fetal growth, we have examined somatomedin synthesis by liver explants established from term fetal rats and maintained in short term organ cultures in chemically defined serum-free media (21). In this report, we demonstrate that fetal liver explants synthesize and release polypeptides biochemically and immunologically related to the well characterized somatomedin, BRL-MSA.  (8,9)." Three broad regions of activity in the chick embryo fibroblast thymidine incorporation assay were identified between K,, = 0.17 and K,, = 0.50, and have been designated "I" (closest to the void), "II" and "III" (most included). Within these regions, seven biologically active MSA species have been resolved by analytical disc gel electrophoresis (pH 2.7, 9 M urea, 12.5% acrylamide): one in Peak I, four in Peak II (II-l, 2, 3, 4), and two in Peak III (III-l, 2). Three of these species have been purified to homogeneity by preparative polyacrylamide electrophoresis:

Cultures
BRL-MSA II-l, an M, = 8,706 single chain polypeptide, and BRL-MSA III-1 and 111-2, single chain polypeptides of M, = 7, 006 (8, 9)." The activities of Sephadex Peak II, Sephadex Peak III, and of the homogeneous components (II-l, III-l, 111.2) are essentially identical in the thymidine incorporation bioassay. The unlabeled BRL-MSA reference preparation used in these studies was a pool of Sephadex Peak II (and included Peptides II-l, 2, 3, and 4).

Assays for BRL-MSA
Rat Liver Membrane Radioreceptor Assay-BRL-MSA II-1 was labeled with Na'""1 using the modified chloramine-T procedure previously described (23). The specific radioactivity of the preparations used in these studies was 90 to 190 Ci/g. Plasma membranes were purified from 100-g Sprague-Dawley rats as previously described (24) and were provided by J. Boone (National Institutes of Health fractions were assayed in the highly specific radioreceptor assay for MSA using rat liver plasma membranes (Fig. 1).  The inhibitory activity in Fractions 25 to 40 was presumptively attributed to fetal MSA. A marker of '""I-labeled BRL-MSA II-1 appeared in Fractions 25 to 32 (Fig. 1). Fractions 33 to 40 probably correspond to lower molecular weight forms of MSA. No somatomedin-binding activity could be detected by direct assay in Fractions 25 to 40 (Table I). Moreover, lz51-MSA remained intact (judged by Sephadex gel filtration) after incubation with Fractions 25 to 40 and liver membranes (data not shown), indicating that the inhibition of binding in the receptor assay did not result from proteolytic destruction of ""I-MSA tracer. Pooled fractions equivalent to Fractions 25 to 40 of the column shown in Fig. 1 have been used in the ensuing studies and are referred to as Sephadex G-75 purified fetal MSA. Sephadex G-75 purified fetal MSA and Sephadex Peak II BRL-MSA inhibited the binding of 12"1-labeled BRL-MSA II-1 to the MSA receptors of rat liver plasma membranes to the same extent and with parallel dose-response curves (Fig. 2) is indicated by the shaded area. The MSA content in the Sephadex G-75 fetal MSA preparation was estimated by the MSA radioreceptor assay (Fig. 2) and is expressed as BRL-MSA equivalents. Sephadex G-75-purified fetal MSA using the rat liver membrane MSA receptor assay and a Sephadex Peak II BRL-MSA standard in six-point dose-response curves as shown in Fig. 2. Unless otherwise noted, in the assays used to quantitate fetal MSA, 50% inhibition of binding of ""I-labeled BRL-MSA was produced by 8 * 2 rig/ml of Peak II BRL-MSA.
It should be emphasized that fetal MSA as determined by the MSA receptor assay constitutes only approximately 5% of the total protein in the Sephadex G-75-purified fetal MSA preparations.
Media collected daily from the first through the 4th days after establishing explants in culture have been used as the source of fetal MSA for purification on Sephadex G-75. As seen in Table II, (Fig. 3). The dose-response curves for BRL-MSA (micrograms of peptide) or fetal MSA (based on apparent MSA content determined in the liver membrane receptor assay) were identical.
Fetal MSA (5 pg/ml) also stimulated the multiplication of serum-starved chick embryo fibroblasts (Table III). The magnitude of the stimulation at 96 h was similar to that obtained with 2% fetal calf serum, and slightly less than that seen with 2.5 pg/ml of BRL-MSA.

Insulin-like
Activity-Sephadex G-75-purified fetal MSA exhibited weak insulin-like activity in the glucose oxidation bioassay in isolated rat adipocytes (Fig. 4). Two preparations of fetal MSA (10-6, 11-17) exhibited activities of 14 and 94 milliunits/mg contrasted with 47 milliunits/mg for BRL-MSA and 25 units/mg for porcine insulin in the same assay. Thus, the insulin-like potency of fetal MSA (0.06 to 0.38% that of insulin) is similar to that of BRL-MSA (0.19% as potent as insulin).
The weak insulin-like activity of fetal MSA was seen not only in the adipocyte bioassay, but also in its weak inhibition of '""I-labeled porcine insulin binding to the insulin receptors of the human lymphoblastoid cell line, IM-9 (Fig. 5). This cell line has abundant, well characterized insulin receptors (35) and does not possess MSA receptors (23,34). Insulins of different species and insulin derivatives inhibit '""I-labeled insulin binding to the IM-9 insulin receptor in proportion to their ability to stimulate glucose oxidation in adipose tissue. In the experiment shown, BRL-MSA was 0.74% as potent an inhibitor of '""I-labeled insulin binding as porcine insulin; fetal MSA also gave weak (0.09% as potent as porcine insulin) but significant inhibition.
Sulfate Incorporation in Hypox Rat Costa1 Cartilage-Fetal MSA, BRL-MSA, and normal rat serum stimulated sulfate uptake in costal cartilage from hypox rats (Table IV). Significant stimulation was seen with the lowest concentrations tested, 0.2 pg/ml of BRL-MSA and 0.27 pg/ml of fetal MSA. Although stimulation by the lowest concentrations of fetal MSA appeared to be greater than that produced by BRL-MSA, formal estimation of potency was not possible since the full dose-response lines were not parallel.

Reactivity of Fetal MSA in Competitive Binding Assays
Fetal MSA cross-reacts in three highly specific competitive binding assays developed for BRL-MSA using ""I-labeled BRL-MSA II-1 as radioligand and rat liver membrane receptors (Fig. 2), rat serum somatomedin-binding proteins (Fig. 6 or rabbit anti-BRL-MSA (Fig. 7) as acceptors. The relative activities of BRL-MSA and fetal MSA in the binding protein assay and the receptor assay were identical (Fig. 6). Fig. 7 demonstrates that fetal MSA is immunologically related to BRL-MSA.
From a logit-log plot of the competitive binding data obtained in the BRL-MSA immunoassay, it can be seen that fetal MSA was nearly parallel to the BRL-MSA Sephadex Peak II standard curve and one-third as potent (Fig. 7). The slight non-parallelism may result from the presence of fetal MSA species corresponding to Peak III BRL-MSA.

Molecular Weight
The molecular weight of Sephadex G-75-purified fetal MSA was estimated by electrophoresis in SDS-17% acrylamide gels (Fig. 8). Fetal MSA was localized by slicing gels, eluting the slices. and assaying the eluates in the liver membrane receptor The positions of known BRL-MSA peptides (I,  The eluates from Slices 27 to 40 were examined in a second immunoassay using assay. Fetal MSA activity, equivalent to 25% of the applied activity, was recovered in the portion of the gel bounded by cytochrome c (Mr = 12,500) and insulin A chain (iV& = 2,200), and including BRL-MSA peptide II-1 (Mr = 8,700).
Growth-promoting Activity, Receptor Reactivity, and Immunoreactivity Are Associated with the Same Peptides in Sephadex G-75-purified Fetal MSA Since fetal MSA constitutes only 5% of the total protein in the Sephadex G-75 preparation, it remained to be demonstrated that the properties previously described were associated with the same components. To test this point, Sephadex G-75 fetal MSA was examined by disc acrylamide electrophoresis at pH 2.7 in 9 M urea (Fig. 9). Portions of the same slab gel were: 1) stained for protein; 2) eluted and assayed in the thymidine incorporation bioassay, the MSA receptor assay, and the MSA radioimmunoassay.
Activity in all three assays was observed in the same region of the gel (Slices 27 to 41) and in two superimposable peaks: one in Fractions 30 to 31, and one in Fractions 36 to 41. The Sephadex G-75 preparation contains many proteins that migrate considerably more slowly than the regions with activity. The major protein band in Fractions 28 to 29 does not coincide with the peak of activity (Fractions 30 to 31). A mixture of BRL-MSA species (Peaks I, II-l, 2, 3, 4, and III-l, 2) run in parallel and stained for protein shows that the activity peaks in the fetal MSA preparation correspond most closely to BRL-MSA II-2 and 111-2. While this does not establish the identity of fetal MSA species and BRL-MSA peptides, it provides direct evidence that three independent properties of the partially purified preparation of fetal MSA are associated with the same molecular species.  (Table II). To examine this more directly, we compared the effect of incubating explants for 24 h with 5 X 10m5 M cycloheximide, an inhibitor of protein synthesis, on albumin, Wfetoprotein, and fetal MSA synthesis (Table V). The concentration of all three proteins in the media was inhibited by 80 to 95%.

DISCUSSION
The family of hormones known as somatomedins or insulinlike growth factors includes four polypeptides purified from human plasma (IGF I, IGF II, somatomedin A, and somatomedin C) and a fifth polypeptide BRL-MSA purified from media conditioned by a rat liver cell line. The present report demonstrates that explants of fetal rat liver maintained in short term organ culture synthesize polypeptides with the properties of somatomedins. These polypeptides have been partially purified by gel filtration and are designated "Fetal MSA" because of their close resemblance to BRL-MSA.
Fetal MSA, like BRL-MSA, stimulated DNA synthesis in and multiplication of chick embryo fibroblasts (Fig. 3, Table III); had weak insulin-like activity in vitro (Figs. 4 and 5); and stimulated ""SO4 incorporation in hypox rat cartilage in vitro (Table  IV). Like BRL-MSA, fetal MSA was stable to acid ( Fig. 1) and to the denaturing agents SDS and urea (Figs. 8 and 9). The size of fetal MSA was estimated as 4,500 to 12,500 by SDS-acrylamide gel electrophoresis (Fig. 8), compared with 8,700 and 7,000 for BRL-MSA II-1 and 111-2, respectively. Fetal MSA showed remarkable reactivity in competitive binding assays developed for BRL-MSA and using MSA receptors, somatomedin-binding proteins or antibodies to BRL-MSA, three macromolecules that selectively recognize different structural features of BRL-MSA and human somatomedins. Receptor reactivity, immunoreactivity and biological activity appeared to be associated with the same molecular species in the partially purified fetal MSA preparation (Fig. 9). Fetal MSA appeared to be synthesized de nouo by fetal liver explants. Serum contamination was not a problem, since the cultures were established and maintained in medium without serum. Release of polypeptides synthesized in uiuo was unlikely, since the content of fetal MSA in media/24 h was relatively constant over 4 days in culture (Table II), constituting 0.2% (Table V) to 0.8% (Table II) of the total secreted protein. More directly, incubation with the protein synthesis inhibitor, cycloheximide, at 5 X lo-" M, decreased the media concentration of fetal MSA, albumin, and cu,-fetoprotein to similar extents (80 to 95%), consistent with rapid synthesis and little storage (Table V).
Although it has been widely assumed that liver is a major site of somatomedin synthesis (1,43), the demonstration that short term liver explants synthesize fetal MSA provides one of the most direct experimental supports for this thesis. Somatomedin activity has been found in plasma and other extracellular fluids, but it is not concentrated in any organ (43). Somatomedin levels are decreased following hepatectomy (44) and in chronic liver disease (45-47). Somatomedin is released from liver perfused with growth hormone, but these studies do not exclude the possibility that liver is merely a repository of somatomedin synthesized elsewhere (48-50). Heinrich et al. (50) have reported that the protein synthesis inhibitor puromycin significantly decreased somatomedin release by liver per&sates. The BRL 3A cell line does synthesize MSA, and cycloheximide does inhibit this synthesis (29).
However, although this cell line was established from the liver of a normal 5-week-old female rat, by virtue of its prolonged life and selection in culture, it has significantly departed from being a normal hepatocyte. For example, BRL 3A cells have lost the ability to synthesize hepatocyte-specific proteins such as albumin." By contrast, fetal liver organ cultures have been shown to preserve significant biochemical features of fetal liver in utero at the developmental stage that the culture is established and provide a much closer reflection of the in uiuo state. Inducible intracellular enzymes such as tyrosine aminotransferase and P-enolpyruvate carboxykinase show the same pattern of hormonal regulation in utero and in explants (51). Similarly, the developmental induction of hepatic glycogen synthetase in explants mimics the in utero pattern. Glycogen synthetase is absent at Day 16 in utero. Glucocorticoids induce the appearance of glycogen synthetase with an identical time course following injection of hypox fetal rats in utero (52), or in vitro administration to liver explants established from 16-day fetuses (53). In addition, fetal liver explants synthesize albumin, ai-fetoprotein, and transferrin (21). The ratio of albumin to a,-fetoprotein is appropriate for the gestational age of the fetus, and is constant for 4 days in culture (21,54). In sum, since fetal liver explants faithfully reflect the biochemical and developmental state of the fetal liver, the synthesis of fetal MSA by liver explants in culture strongly implies that normal fetal liver in utero synthesizes MSA.
Synthesis of somatomedin by fetal liver explants is particularly intriguing because several recent observations have suggested that somatomedins may be important in fetal growth. Low but detectable levels of somatomedin have been found in human cord blood by several investigators using a variety of assays: e.g. sulfation in cartilage of rat (15, 19) and pig (12, 17), radioreceptor assays for somatomedin A (18) and somatomedin C (14), competitive binding protein assay for IGF-I (20), and somatomedin C radioimmunoassay (16). Somatomedins also are present in amniotic fluid (13,18). Positive correlations of somatomedin levels in human cord blood determined by bioassay (55), competitive binding protein assay (56) and radioimmunoassay (57) with birth weight (55-57) and length (55) have been reported in preliminary form. Abundant somatomedin receptors are present in membranes prepared from different tissues of fetal pigs at different gestational ages (14), suggesting that the fetus does have the capacity to respond to somatomedins.
Although Stuart et al. (58) were unable to detect somatomedin activity in blood from term rat fetuses using a rat cartilage bioassay, we recently have demonstrated a 20-to loo-fold increase of immunoreactive MSA in fetal rat serum (19-day gestation) over the corresponding maternal samples (29).7 Moreover, preliminary results suggest that among the presumed multiple somatomedins in rat blood, the component immunologically related to BRL-MSA may be selectively increased in fetal blood. Little is known of the regulation of fetal somatomedins. Growth hormone does not appear to be a major factor. Fetal growth hormone levels are high and constant, and do not correlate with fetal somatomedin levels (11,12,17,18). Recent reports suggest that endogenous (59) or exogenous (60) placental lactogens may induce somatomedin synthesis in hypox rats, and that human placental lactogen and radioimmunoassayable somatomedin C levels in maternal serum are closely correlated during pregnancy (57). Insulin per se or hyperglycemia deserve consideration as possible inducers of fetal somatomedin synthesis. Insulin caused release of somatomedins ' H. G. Coon, personal communication. '