Nutrition and Somatomedin XXV. Regulation of Insulinlike Growth Factor Binding Protein 1 in Primary Cultures of Normal Rat Hepatocytes BETTY C. VILLAFUERTE, STEVEN GOLDSTEIN, LIAM J. MURPHY, AND LAWRENCE S. PHILLIPS

Although mRNAs encoding insulinlike growth factor (IGF) binding proteins (BPs) are present in adult rat liver and IGF BP-1 circulates at elevated levels in diabetic animals, there is little knowledge of the metabolic regulation of IGF BPs in normal tissues. We examined the release of IGF BPs by adult rat hepatocytes maintained in primary culture. When cultured for 2 days in the absence of added insulin, hepatocytes released a BP identified as BP-1 on the basis of ~30,000-/Wr on ligand blotting and reactivity with antiserum to human BP-1 in immunoblotting and immunoprecipitation studies. Release of BP-1 was sensitive to insulin with suppression of 24 ± 4, 73 ± 5, and 64 ± 14% at 1 0 1 0 , 1 0 8 , and 1 0 6 M insulin, respectively; ED50 was —1.7 x 10~9 M, which is within the physiological range. Suppression by insulin was reversible and began within 3 h. Because normal hepatocytes in primary culture exhibit insulinresponsive release of both BP-1 and IGF-I, this system may be an ideal model for studies of molecular mechanisms of metabolic regulation. Diabetes 40:83741, 1991

T

he insulinlike growth factors (IGFs) are structurally similar to proinsulin, exhibit anabolic and mitogenic effects in vivo and in vitro, and circulate largely bound to carrier proteins (1). Three immunologically distinct IGF binding proteins (BPs) have been identified: BP1, 28,000-30,000 Mr (2); BP-2, a 30,000-/Wr fetal form (3); and BP-3, an ~29,000-Mr molecule glycosylated to -53,000 M, (4). BP-3 is saturated and fluctuates sluggishly under

From the Division of Endocrinology and Metabolism, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and the Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Canada. Address correspondence and reprint requests to Lawrence S. Phillips, MD, Department of Medicine, Emory University School of Medicine, 69 Butler Street, SE, Atlanta, Georgia 30303. Received for publication 30 September 1990 and accepted in revised form 19 February 1991.

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physiological conditions; diabetes tends to lower BP-3 (5). In contrast, BP-1 is unsaturated and varies rapidly, 4-fold under normal conditions and up to 10- to 20-fold in disease; diabetes and nutritional deprivation raise BP-1 (6,7). In vivo studies have revealed diurnal variation of BP-1 (8), increased secretion during the counterregulatory response to insulininduced hypoglycemia (9), and suppression by oral glucose loads and hyperinsulinemia during euglycemic clamps (7). However, although release of BP-1 has been investigated in human liver cell lines (10-12), relatively little is known about the secretion of BP-1 by normal cells from adult animals, which is needed to study underlying mechanisms of hormonal regulation. In this study, we report the use of normal rat hepatocytes in primary culture as a model for examining BP-1 production and its physiological regulation by insulin. RESEARCH DESIGN AND METHODS

Recombinant IGF-I was purchased from AMGen Biologicals (Thousand Oaks, CA) and iodinated by lactoperoxidase to 300 Ci/g. Human BP-1 purified from placenta (PP12) and polyclonal rabbit antiserum against hBP-1 were generously provided by H. Bohn (Behringwerke, Germany) and A.-M. Suikkari (Helsinki, Finland). BRL 3A-conditioned medium and rabbit antiserum against rBP-2 were gifts from M. Rechler (Bethesda, MD), and human insulin was a gift from Lilly (Indianapolis, IN). Staphylococcus aureus cells (Pansorbin) were purchased from Calbiochem (La Jolla, CA), tissue-culture media was from JRH Biosciences (Lenexa, KS), goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) was from Kirkegaard and Perry (Gaithersburg, MD), and all other chemicals were from Sigma (St. Louis, MO). Serum was obtained from nondiabetic male Sprague-Dawley rats (-150 g) and diabetic rats (2 days after - 3 0 0 mg/kg i.v. streptozocin). Hepatocyte culture. Hepatocytes were isolated from nondiabetic 150- to 200-g male Sprague-Dawley rats with a twostage collagenase perfusion technique (13). An estimated 3.6 x 106 viable cells were incubated in 60-mm collagencoated dishes at 37°C in 5% CO2/95% air. Initially they were

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IGF BP-1 IN HEPATOCYTE CULTURE

incubated for 3 h in Dulbecco's modified Eagle's medium and F-12 containing 10% fetal calf serum until cells attached, and then they were maintained with daily changes of serumfree medium with insulin concentrations as indicated. Ligand blot analysis. With the technique of Hossenlopp et al. (14), conditioned medium was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under nonreducing conditions, and proteins were transferred to nitrocellulose and blocked sequentially with 3% Nonidet P-40,3% bovine serum albumin (BSA), and 0.1% Tween 20. BPs were identified by incubation with 125l-labeled IGF-I (106 counts/min[cpm]) overnight at room temperature followed by autoradiography for 24 h, and binding patterns were quantitated by laser densitometry. Immunoblotting. After electrophoresis and transfer as described above, membranes were blocked with 7% nonfat dry milk, then incubated with antiserum to hBP-1 (1:500) or rBP-2 (1:200) for 4 h, washed, and incubated overnight with HRP-linked goat anti-rabbit IgG followed by 4-chloro-1naphthol with added 30% H2O2. Immunoprecipitation. As modified from Yang et al. (15), samples were incubated with 125I—IGF-I (4 x 105 cpm) in 10 mM Tris and 0.5% BSA, pH 7.4, for 4 h at 4°C to form 125 I—IGF-I—BP complexes. Unbound tracer was removed with 1% activated charcoal, and an aliquot (1/3o) of the charcoal supernatant was incubated overnight at 4°C with antiserum to BP-1 or BP-2. Antibody-BP-125l-IGF-l complexes were precipitated with 1% Pansorbin. Analysis of variance and t tests were employed when comparing multiple groups. Results were considered statistically significant when two-tailed P < 0.05. RESULTS Identification of BP-1 in hepatocyte primary culture. IGF BPs in hepatocyte-conditioned medium were delineated by

Serum vs Cells

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4630-

PP12 HEP-

PP12 NRS

0

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FIG. 1. Ligand blotting of insulinlike growth factor (IGF) binding proteins (BPs) in rat serum and hepatocyte-conditioned medium. Samples were electrophoresed under nonreducing conditions, electroblotted to nitrocellulose, incubated with 125l-labeled IGF-I, and developed by autoradiography. Left: PP 12 ,100 ng human BP-1 {left); NRS, 3 JJLI normal adult rat serum; DRS, 1 fxl diabetic rat serum; HEP, 7.5 |il primary rat hepatocyte-conditioned medium. Right: 200 ng PP12 and 10-jxl aliquots (right) of conditioned medium from hepatocytes cultured without and with added insulin.

838

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FIG. 2. Identification of hepatocyte insulinlike growth factor (IGF) binding proteins (BPs) by immunoblotting as described in METHODS. Left: PP,2,100 ng human BP-1; HEP, 20 |xl hepatocyte-conditioned medium; 50 yA BRL 3A-conditioned medium; and DRS, 20 \x,\ diabetic rat serum tested with antiserum to hBP-1 (1 500 dilution). Right: 200 ng PP12, 20 \i\ HEP, 20 jil DRS, and 50 p.l BRL 3A-conditioned medium tested with anti-rBP2 (1:200 dilution).

ligand blotting and compared to hBP-1 and BPs in normal adult rat and diabetic rat serum (Fig. 1, left). BP-1 extracted from human decidua appeared at 28,000 Mn and the predominant BP in normal rat serum appeared as an -46,000M, cluster. BPs of -30,000 Mr were markedly elevated in diabetic rat serum but were barely detectable in normal rat serum. Hepatocyte-conditioned medium contained an ~30,000-Mr IGF BP similar in size to BPs detected in diabetic rat serum and slightly higher in apparent molecular weight than hBP-1. The type of BP produced by adult rat hepatocytes was then identified according to reactivity with antibodies to hBP1 and rBP-2. Western blots revealed that antiserum to hBP1 recognized the BP extracted from human decidua, hepatocyte-conditioned medium, and diabetic rat serum but did not recognize the BP in medium conditioned by BRL 3A cells (Fig. 2). In contrast, antiserum to rBP-2 did not recognize BPs from human decidua, hepatocyte-conditioned medium, or diabetic rat serum but recognized the —30,000M, BP in BRL 3A cells, previously identified as rBP-2 (15). BPs produced by human decidua, diabetic rats, and rat hepatocytes were immunoprecipitated only by antiserum to hBP-1, whereas the BP produced by BRL 3A cells was immunoprecipitated only by antiserum to rBP-2 (Fig. 3). In combination, these findings indicate that the predominant BP secreted by adult rat hepatocytes is BP-1, similar to BP-1 found in diabetic rat serum (16) and immunologically related to hBP-1 but distinct from BP-2 produced by fetal and neonatal rat liver. In separate studies, we found RNA from cultured hepatocytes to be recognized by cDNA for rBP-1 (Fig. 4) but not by cDNA for rBP-2 (data not shown). Insulin regulation of BP-1 secretion in rat hepatocyte primary culture. To evaluate the effect of insulin on BP-1 release by cultured rat hepatocytes, medium was supplemented with insulin, changed daily, and harvested after 3 days. In a representative experiment, increasing concentra-

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B.C. VILLAFUERTE AND ASSOCIATES FIG. 3. Identification of hepatocyte binding protein (BP) by immunoprecipitation. Samples were incubated with 125l-labeled insulinlike growth factor I (IGF-I), and unbound tracer was removed with activated charcoal. The charcoal supernatant contained 24-58% of input radioactivity, in contrast to 0.9% in the absence of BPs. Aliquots of charcoal supernatant were incubated with anti-hBP-1 or rBP-2 and precipitated with Pansorbin, and pellet radioactivity was quantitated. PP12, 200 ng human BP-1 (O); DRS, 60 JJLI diabetic rat serum (A), 200 \L\ BRL 3A-cortditioned medium (A), and 60 fxl rat hepatocyte-conditioned medium ( • ) .

1:160001:8000 1:4000 1:2000 1:1000 Antibody dilution

tions of insulin led to progressive suppression of BP-1 release (Fig. 1, right). Suppression was 24 ± 4% at 10~10 M insulin (P = 0.17) and 73 ± 5 and 64 ± 14% at 10' 8 and 10" 6 M insulin, respectively (both P < 0.05 vs. 0 insulin); there was no difference in BP-1 release between 10~8 and 10" 6 M insulin (Fig. 5). To confirm the regulatory effect of insulin on rBP-1 release, hepatocytes were cultured with insulin for 2 days and then switched to lower or higher concentrations for another 2 days; BP-1 release was compared to that in cultures maintained at the same concentration of insulin for 4 days. There was a 149% increase in BP-1 release when medium was switched from high to low insulin (10" 6 to 10~10 M, P < 0.05), yet there was no significant change when cells were cultured in high insulin for 4 days (P = 0.25; Fig. 6). Conversely, there was a 56% reduction in BP-1 release when medium was switched from low to high insulin (10"1° to 10- 6 M, P < 0.05) with no change when cells were maintained in low insulin for 4 days. In combination, these findings revealed maximal suppression of BP-1 by insulin concentrations within the physiological range and showed the inhibitory effect of insulin to be reversible. DISCUSSION

Human BP-1 has been isolated from amniotic fluid, placenta, and medium conditioned by HepG2 cells. Human BP-1 cDNA encodes an ~26,000-Mr protein rich in cysteines, with an NH2-terminal similar to that of other IGF BPs (2,7,17,18). Less is known regarding rat BP-1; a cDNA isolated from a rat decidual cDNA library encodes a protein with a similar NH2-terminal (19), and Unterman et al. (20) have recently

1:160001:8000 1:4000 1:2000 Antibody dilution D — D PP12 • — • Hepotoeyte A — A 0RS A—ABRL 3A

purified a BP from H-4-II-E cell-conditioned medium that appears to be identical to rat decidual BP-1. The biological actions of BP-1 appear to include both inhibition (21) and facilitation (22) of IGF action. Regulation of BP-1 is insulinsensitive in humans (6), but underlying mechanisms are difficult to dissect in vivo because of concomitant changes in other hormones and metabolism of metabolic fuels. In vitro studies of BP-1 regulation indicate suppression by insulin; to date, examinations have been limited to HepG2 cells, an "immortal" human liver cell line (10-12), and human fetal liver explants in short-term culture (23). In this study, primary cultures of hepatocytes from normal adult rats appeared to produce BP-1 as the predominant IGF BP. Although BP-1 mRNA is expressed in adult rat liver (19), and rBP-1 circulates at elevated levels in diabetic rats (16), BP-1 production by cultured rat hepatocytes has not previously been demonstrated. In our system, BP-1 was produced in high abundance, 4-40 n,g/ml in 24 h (based on densitometric analysis vs. PP12). Others have reported BP-1 production by human HepG2 hepatoma cells (10-12,24), rat H35 hepatoma cells (15), and human fetal liver explants (23). Although liver cell lines exhibit some characteristics of normal adult liver, e.g., albumin synthesis (25), full normal physiology may not be represented because these cells are typically deficient in transcription factors (26) and insulinresponsive enzymes, i.e., glucokinase (D. Granner, unpub-

100--

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FIG. 4. Northern blot of insulinlike growth factor (IGF) binding protein (BP) 1 mRNA in normal rat liver and separate preparations of normal rat hepatocytes cultured for 2 days without added insulin. Total RNA (20 ng/sample) was isolated from liver (28) and hepatocytes (29), separated on a 1.2% agarose formaldehyde gel, transferred to charged nylon, hybridized with a 32P-labeled cDNA probe for rat decidual BP-1 (19), and visualized by autoradiography after 3 days exposure (30).

DIABETES, VOL. 40,'JULY 1991

••

10" l u 10 - 8 [Insulin], M

10

-6

FIG. 5. Effect of insulin on hepatocyte binding protein (BP) 1 production. Densitometric analysis of ligand blots of medium conditioned by rat hepatocytes cultured without and with added insulin. Results were normalized to percent of control medium (without insulin, n = 4). Values are means ± SE.

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IGF BP-1 IN HEPATOCYTE CULTURE

ACKNOWLEDGMENTS

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DAY OF HEPATOCYTE CULTURE FIG. 6. Effect of change in insulin on binding protein (BP) 1 secretion by hepatocytes. Left: densitometric analysis of ligand blots assessing BP-1 produced by normal rat hepatocytes when cells were maintained in 10~6 M insulin for 2 days ( • , day 2) then either continued in 10~6 M insulin ( • , day 4) for another 2 days or switched to 10~10 M insulin (O, day 4). Right: BP-1 produced by hepatocytes initially exposed to 10" 10 M insulin for 2 days ( • , day 2), then either continued in 10" 10 M insulin ( • , day 4) or switched to 10~6 M insulin (O, day 4); n = 3 each. Values are means ± SE. Error bar (not visible) is contained within datum symbol.

lished observations). In contrast, monolayer cultures of normal hepatocytes exhibit a wide variety of metabolic and secretory functions including insulin-sensitive release of IGFI, which is characteristic of intact liver (13). We have also demonstrated that rat hepatocyte secretion of BP-1 is sensitive to insulin, which is consistent with in vivo findings of suppression of BP-1 release by insulin during euglycemic clamps (7). Maximum suppression was found with 10" 8 M insulin with ED50 estimated to be 1.7 x 10" 9 M. This is consistent with in vivo findings of suppression of BP1 release within the physiological range of insulin concentration in the portal circulation and close to 10-fold more sensitive to insulin than HepG2 cells (10,11). In separate studies (data not shown), we have found suppression of BP1 rriRNA within 90 min after addition of insulin, suggesting rapid regulation of hepatocyte production of BP-1. This is similar to the decline in hBP-1 seen in 2-3 h with insulin infusion (7). Although Conover and Lee (12) have shown that insulin acts through its own receptor to inhibit BP-1 secretion, the postreceptor mechanisms involved have not been elucidated. Baxter and Lewitt (23) have suggested that intracellular availability of glucose may be critical in the control of BP-1 synthesis. The normal adult rat liver cell appears to be unique in exhibiting metabolically regulated synthesis of both BP-1 and IGF-I. Although BP-1 mRNA is found in a wide variety of tissues, levels of IGF-I mRNA are 50-100 times higher in the liver than in other tissues (27). Because release of both BP-1 and IGF-I by normal rat hepatocytes in primary culture is sensitive to insulin at physiological concentrations, this system may constitute an ideal model in which to examine molecular mechanisms of nutritional/hormonal regulation of these export proteins.

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This work was supported in part by Grants DK-33475 and DK-41187 from the National Institutes of Health. We thank Yueming Wang for assistance with hepatocyte culture and Kathy Mullen and Mary Lou Mojonnier for assistance in preparation of the manuscript.

1. Daughaday WH, Rotwein P: Insulin-like growth factors I and II: peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 10:68-91, 1989 2. Brewer MT, Stetler GL, Squires CH, Thompson RC, Busby WH, Clemmons DR: Cloning, characterization, and expression of a human insulin-like growth factor binding protein. Biochem Biophys Res Commun 152:128997,1988 3. Brown AL, Chiariotti L, Orlowski CC, Mehlman T, Burgess WH, Ackerman EJ, Bruni CB, Rechler MM: Nucleotide sequence and expression of a cDNA clone encoding a fetal rat binding protein for insulin-like growth factors. J Biol Chem 264:5148-54, 1989 4. Baxter RC, Martin JL, Beniac VA: High molecular weight insulin-like growth factor binding protein complex: purification and properties of the acidlabile subunit from human serum. J Biol Chem 264:11843-48, 1989 5. Baxter RC, Martin JL: Radioimmunoassay of growth hormone-dependent insulinlike growth factor binding protein in human plasma. J Clin Invest 78:1504-12, 1986 6. Cotterill AM, Cowell CT, Baxter RC, McNeil D, Silinik M: Regulation of the growth hormone-independent growth factor-binding protein in children. J Clin Endocrinol Metab 67:882-87, 1988 7. Suikkari A-M, Koivisto VA, Rutanen E-M, Yki-Jarvinen H, Karonen S-L, Seppala M: Insulin regulates the serum levels of low molecular weight insulin-like growth factor-binding protein. J Clin Endocrinol Metab 66:266-72, 1988 8. Baxter RC, Cowell CT: Diurnal rhythm of growth hormone-independent binding protein for insulin-like growth factors in human plasma. J Clin Endocrinol Metab 65:432-40, 1987 9. Yeoh SI, Baxter RC: Metabolic regulation of the growth hormone independent insulin-like growth factor binding protein in human plasma. Acta Endocrinol 119:465-73, 1988 10. Cotterill AM, Cowell CT, Silink M: Insulin and variation in glucose levels modify the secretion rates of the growth hormone-independent insulinlike growth factor binding protein-1 in the human hepatoblastoma cell line HepG2. J Endocrinol 123:R17-20, 1989 11. Powell DR, Suwanichkul A, Cubbage M, Lee PDK: Regulation of insulinlike growth factor binding protein-1 (IGFBP-1) protein levels, mRNA levels and promoter activity by insulin (IN) and IGF-1 in HepG2 (Abstract). Endocr Soc Aust Proc 280A, 1990 12. Conover CA, Lee PDK: Insulin regulation of insulin-like growth factorbinding protein production in cultured HepG2 cells. J Clin Endocrinol Metab 70:1062-67, 1990 13. Goldstein S, Pao C-l, Wang Y, Alam S, Farmer PK, Sertich G, Phillips LS: Molecular regulation of IGF-1 by insulin in normal rat hepatocytes in culture (Abstract). Diabetes 39 (Suppl. 1):141A, 1990 14. Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M: Analysis of serum insulin-like growth factor binding proteins using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138-43, 1986 15. Yang YW-H, Brown AL, Orlowski CC, Graham DE, Tseng LY-H, Romanus JA, Rechler MM: Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rlGFBP-1,2, or 3. MolEndocrinol 4:29-38, 1990 16. Unterman TG, Oehler DT, Becker RE: Identification of a type I insulin-like growth factor binding protein (IGF BP) in serum from rats with diabetes mellitus. Biochem Biophys Res Commun 163:882-87, 1989 17. Lee Y-L, Hintz RL, James PM, Lee PDK, Shively JE, Powell DR: Insulinlike growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HepG2 hepatoma cells: predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors. Mol Endocrinol 2:404-11, 1988 18. Cianfarani S, Holly JMP: Somatomedin-binding proteins: what role do they play in the growth process? Eur J Pediatr 149:76-79, 1989 19. Murphy LJ, Seneviratne C, Ballejo G, Croze F, Kennedy TG: Identification and characterization of a rat decidual insulin-like growth factor-binding protein complementary DNA. Mol Endocrinol 4:329-36, 1990 20. Unterman TG, Oehler DT, Gotway M, Morris PW: Purification and NH2terminal sequence analysis of the rat type 1 insulin-like growth factor binding protein (rlGFBP-1). Endocrinology 127:789-97, 1990 21. Ritvos O, Ranta T, Jaikanen J, Suikkari A-M, Vouitilainen R, Bohn H, Rutanen E-M: Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-1 in cultured choriocarcinoma cells. Endocrinology 122:2150-57, 1988

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B.C. VILLAFUERTE AND ASSOCIATES 22. Elgin RG, Busby WH Jr, Clemmons DR: An insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-1. Proc Natl Acad Sci USA 84:3254-58, 1987 23. Lewitt MS, Baxter RC: Regulation of growth hormone-independent insulinlike growth factor-binding protein (BP-28) in cultured human fetal liver explants. J Clin Endochnol Metab 69:246-52, 1989 24. Powell DR, Lee PDK, Shively JE, Eckenhausen M, Hintz RL: Method for purification of an insulin-like growth factor-binding protein produced by human HepG2 hepatoma cells. J Chromatogr 420:163-70, 1987 25. Cassio D, Weiss MC, Ott M-O, Sala-Trepat JM, Fries J, Erdos T: Expression of the albumin gene in rat hepatoma cells and their dedifferentiated variants. Cell 27:351-58, 1981 26. Friedman AD, Landschulz WH, McKnight SL: CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured

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hepatoma cells. Genes Dev 3:1314-22, 1989 27. Murphy LJ, Bell Gl, Friesen HG: Tissue distribution of insulin-like growth factor I and II messenger ribonucleic acid in the adult rat. Endocrinology 120:1279-82, 1987 28. Goldstein S, Harp JB, Phillips LS: Nutrition and somatomedin. XXII. Molecular regulation of insulin-like growth factor-1 during fasting and refeeding in rats. J Mol Endocrinol. In press 29. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, Cold Spring, NY, Harbor Lab., 1989, p. 7.10-7.11 30. Goldstein S, Sertich G, Levan KR, Phillips LS: Nutrition and somatomedin. XIX. Molecular regulation of insulin-like growth factor-l in streptozotocindiabetic rats. Mol Endocrinol 2:1093-100, 1988

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Nutrition and somatomedin. XXV. Regulation of insulinlike growth factor binding protein 1 in primary cultures of normal rat hepatocytes.

Although mRNAs encoding insulinlike growth factor (IGF) binding proteins (BPs) are present in adult rat liver and IGF BP-1 circulates at elevated leve...
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