Expression and regulation of growth hormone (GH) receptor messenger ribonucleic acid (mRNA) in rat adipose tissue, adipocytes, and adipocyte precursor cells: GH regulation of GH receptor mRNA.

0013-7227/91/1293-1155S03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 3 Printed in U.S.A.

Expression and Regulation of Growth Hormone (GH) Receptor Messenger Ribonucleic Acid (mRNA) in Rat Adipose Tissue, Adipocytes, and Adipocyte Precursor Cells: GH Regulation of GH Receptor mRNA* KERSTIN VIKMAN, BJORN CARLSSON, HAKAN BILLIG, AND STAFFAN EDEN Department of Physiology, University of Goteborg, S-400 33 Goteborg, Sweden

ABSTRACT. The effects of hypophysectomy and hormonal replacement therapy on GH receptor (GH-R) gene expression was studied in rat adipose tissue with a cRNA probe corresponding to the amino-terminal of the hepatic GH-R. Male SpragueDawley rats, 50-65 days of age, were used. In all fat depots tested (epididymal, retroperitoneal, and sc), two transcripts with an estimated size of 4.0 and 1.2 kilobases (kb), respectively, were detected. An intermediate-size transcript (2.6 kb) was sometimes observed. Also, isolated adipocytes and adipocyte precursor cells from the epididymal fat pad expressed these GH-R transcripts. The pituitary dependence of GH-R gene expression was analyzed in epididymal fat. Hypophysectomies were performed at 50 days of age, and the rats were then given replacement therapy with L-T4 (10 >ig/kg-day) and hydrocortisone (400 jig/kgday). Hypophysectomy decreased the abundance of both the 4.0 and the 1.2-kb transcripts, an effect that in part was restored by GH

treatment. A solution hybridization RNase protection assay was then used to further characterize the effect of GH treatment of hypophysectomized rats on GH-R gene expression. A single injection of human GH (100 Mg/rat) increased GH-R mRNA levels within 1 h, and maximal levels were reached between 312 h after the injection. The increase in GH-R mRNA levels was dose dependent and was observed also after prolonged treatment (1 or 5 mg/kgday for 6 days) with bovine GH. These results confirm that GH-R mRNAs are present in rat adipose tissue from different fat depots. GH-R transcripts of the same estimated size were detected in isolated adipocytes and adipocyte precursor cells. Furthermore, the results show that there is a rapid and GH-dependent regulation of GH-R mRNA levels in adipose tissue. (Endocrinology 129: 1155-1161, 1991)

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GH-R cDNA sequence is well conserved between species and encodes a GH-R protein with a single centrally located transmembrane domain. The extracellular domain of the GH-R shares many homologies with the serum GH-binding protein (GH-BP) and has been suggested to be produced by a proteolytic release of the extracellular domain of the GH-R (9). In mice and rats a specific cDNA has been isolated encoding a GH-BP identical to the GH-R, but with the transmembrane and cytoplasmic domains substituted with a hydrophilic tail (10, 11). Transcripts with estimated sizes of the GH-R and GH-BP are present in several tissues in the rat (12), including adipose tissue (13). The present study was undertaken to investigate the expression of GH-R mRNA in different fat depots, isolated adipocytes, and adipocyte precursor cells and to investigate the possible role of GH in regulating the levels of GH-R transcripts.

DIPOSE tissue is a target tissue for several effects of GH. GH is believed to induce its effect in target cells through interaction with membrane-bound receptors. Labeled GH has been shown to specifically bind to adipocytes from rats (1) and man (2). Previous studies using binding techniques have indicated that the GH receptor (GH-R) of adipocytes has a rapid turnover that is dependent upon protein synthesis (3), with a half-life of 30-45 min (4). Moreover, binding studies have indicated that the GH-R in adipocytes is hormonally regulated by thyroid hormones, insulin (5), and GH itself (4, 6). The recent purification of a GH-R from rabbit liver and the subsequent cloning of GH-R cDNAs in several species (7, 8) have opened new possibilities to study the function, structure, and regulation of the GH-R. The Received December 26,1990. Address all correspondence and requests for reprints to: Dr. Staffan Eden, Department of Physiology, University of Goteborg, P.O. Box 33031, S-400 33 Goteborg, Sweden. * This research was supported by the Swedish Medical Research Council (Grant 8269), Nordisk Insulinfond, the Goteborg Medical Society, the Faculty of Medicine, University of Goteborg, and the Swedish Society of Medical Research.

Materials and Methods Animals Male Sprague-Dawley rats, 50-65 days old (Alab Laboratory Services, Stockholm, Sweden), were used. They were kept in

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GH REGULATION OF GH RECEPTOR mRNA

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constant temperature with a 14-h light, 10-h dark cycle. Tap water and pelleted food (type R36, Ewos, Sodertalje, Sweden) were freely available. Hypophysectomy was performed by the standard parapharyngeal approach at 50 days of age under combined ketamine hydrochloride (67 mg/kg; Ketalar, ParkeDavis, Detroit, MI) and xylazine (13 mg/kg; Rompun, Bayer, Lever-Kusen, Germany) anesthesia. Body weight was measured daily, and rats gaining more than 0.5 g/day in body weight during the 1 week observation period were excluded. No rat had to be excluded due to excessive weight loss during the experimental period. Hormonal treatment L-T4 (Nycomed Ltd., Oslo, Norway; 10 ^g/kgday) and hydrocortisone phosphate (Solu-Cortef, Upjohn, Puurs, Belgium; 400 ^g/kg-day) diluted in saline were given as a daily sc injection to hypophysectomized (hx) rats. Recombinant human GH (hGH; Genotropin; 2.7 IU/mg) was generously provided by Kabi AB (Stockholm, Sweden), and recombinant bovine GH (bGH; potency not specified) was generously provided by American Cyanamide Co. (Princeton, NJ). hGH was used in experiments in which the effect of a short period of treatment ( 10 mM dithiothrol, and 25% formamid with a 36S-labeled GHR cRNA probe in a volume of 40 nl. The samples were then treated with 40 ng RNase A and 2 ng RNase Tr in the presence of 100 Mg herring sperm DNA for 45 min at 37 C in a volume of 1 ml. Protected probe was precipitated with 100 jtl trichloroacetic acid (6 M). The precipitate was collected on glass-fiber filters (GF/C, Whatman International Ltd., Maidstone, England) and counted in a scintillation counter. A tissue TNA preparation, originally compared with an in vitro transcribed GH-R mRNA, was used to generate a standard curve. The standard curve was linear between 0.1-16 amol. The DNA content of the samples was analyzed (19), and 10-45 /xg DNA in samples were assayed. Within this range, the hybridization signal parallelled the standard curve. In all assays the lowest concentration of GH-R mRNA measured was well above the lowest point of the standard curve. Each TNA sample was analyzed in duplicate. The results are expressed as the amount of GH-R mRNA per DNA (10~18 mol/Mg). The coefficient of variation was 6%, determined as the mean coefficient of variation of duplicates from three different assays. GH-R probe A pT7T3 18U vector carrying a 560-basepair BamHI fragment (8) was linearized with EcoRl cleaving in the cloning box of the vector, and labeled ([32P]UTP or [35S]UTP) cRNA was generated with T 3 polymerase (Promega, Madison, WI) under the conditions indicated by the manufacturer. Statistics Values are given as the mean ± SE. Comparisons between groups were performed with analysis of variance, followed by Student-Newman-Keuls multiple range test (20). P < 0.05 was considered significant.



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Results A GH-R cRNA probe corresponding to part of the extracellular domain of the GH-R recognized two major transcripts, with estimated sizes of 4.0 and 1.2 kilobases (kb) in RNA extracted from epididymal, retroperitoneal, and sc fat depots (Fig. 1). In addition, a low abundant transcript with an estimated size of 2.6 kb was also evident in some of the RNA preparations. To determine in which cell type the GH-R gene is active, RNA was extracted from adipocytes and adipocyte precursor cells and analyzed for the presence of GH-R transcripts. As can be seen in Fig. 2, both transcripts were present in both cell fractions. Levels of GH-R mRNA were higher in adipocytes than in adipocyte precursor cells. To study hormonal regulation of the abundance of GH-R transcripts, the effects of hypophysectomy and GH administration were investigated. Hypophysectomy resulted in a decrease in both major transcripts in epi-

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I8S ~ FIG. 1. Analysis of GH-R mRNA in different fat depots. Male rats, 40 days old, were killed, and adipose tissues were excised and immediately frozen. Fat depots used were epididymal (Ep), retroperitoneal (Rp), and sc (Sc) depots. Total RNA was prepared from two individual rats. Twenty micrograms of total RNA were electrophoresed, transferred, and hybridized with a 32P-labeled GH-R RNA probe, as described in Materials and Methods. Upper panel, Hybridized GH-R mRNAs; lower panel, ethidiumbromide staining of ribosomal RNA.

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FIG. 2. Analysis of GH-R mRNA in adipocytes and adipocyte precursor cells isolated from epididymal fat pads of normal rats. Twenty micrograms of total RNA were electrophoresed, transferred, and hybridized with a 32P-labeled GH-R RNA probe, as described in Materials and Methods. Upper panel, Hybridized GH-R mRNAs; lower panel, ethidiumbromide staining of ribosomal RNA.

didymal fat (Fig. 3). When hx rats were given a high total dose of hGH (200 /xg, given sc at 4-h intervals in a total of five injections), both major transcripts increased (Fig. 3). To further quantify the effects of hypophysectomy and GH treatment, a solution hybridization assay was used. A single iv injection of hGH (100 fig) resulted in increased GH-R mRNA levels by 1 h after the injection. Levels increased further between 2 and 12 h and thereafter


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FIG. 4. Effect of hypophysectomy and a single iv injection of hGH on GH-R mRNA levels in epididymal fat pads. Male rats were hx at 50 days of age and then given replacement therapy with L-T4 and hydrocortisone. One week after hypophysectomy, 100 jtg hGH were injected iv, and the rats were killed at the times indicated. Sham-operated agematched rats were given an iv injection of saline (Sham). GH-R mRNA levels were determined by a solution hybridization assay, as described in Materials and Methods. There were five or six rats in each group. Vertical bars represent the SE. +, P < 0.01 us. all other groups.

18 S — FIG. 3. Effect of hypophysectomy and GH replacement therapy on GH-R mRNA expression. Male rats were hx (Hx) or sham-operated (Sham) at 50 days of age. One week later hGH (200 jig) was given sc every fourth hour in a total of five injections. Twenty micrograms of total RNA were electrophoresed, transferred, and hybridized with a 32 P-labeled GH-R RNA probe, as described in Materials and Methods. Fat from two rats were pooled. Upper panel, Hybridized GH-R mRNAs; lower panel, ethidiumbromide staining of ribosomal RNA.

decreased, but were still elevated compared to levels before the injection (Fig. 4). When hGH was given as a single sc injection in two different doses (100 and 1000 Hg) 10 h before death, a dose-dependent increase in GHR mRNA levels was observed (Fig. 5). In all of these experiments GH could only partly reverse the decrease in the levels of GH-R transcripts seen after hypophysectomy. In the next series of experiments the effect of prolonged treatment of hx rats with GH was investigated. bGH was given as two daily sc injections at 12-h intervals in two different doses (1 or 5 mg/kg-day) for 6 days. The animals were killed at different times after the last injection, and GH-R mRNA levels were determined in epididymal fat pads by solution hybridization assay. The time course for the effect of GH was similar regardless of the dose given, resulting in increased levels of GH-R mRNA 2 h and maximal levels 6 h after hormone administration. After 12 h, levels in epididymal fat pads of animals given the lower dose of bGH were as low as

Sham

Hx

100

1000

FIG. 5. Effect of hypophysectomy and a single sc injection of hGH (100 or 1000 ng) on GH-R mRNA levels in epididymal fat pads. Male rats were hypophysectomized (Hx) or sham-operated (Sham) at 50 days of age and then given replacement therapy with L-T4 and hydrocortisone. One week after hypophysectomy, hGH (100 or 1000 ng) or saline was injected sc. The rats were killed 10 h after the injection. GH-R mRNA levels were determined in epididymal fat pads by a solution hybridization assay, as described in Materials and Methods. There were six rats in each group, with one observation from each rat. Vertical bars represent the SE. +,P< 0.01 us. all other groups.

those in tissues from untreated hx animals, whereas levels in animals treated with the higher dose were still elevated. Again, in all hx animals regardless of treatment, GH-R mRNA levels were lower compared to those in normal animals (Fig. 6). Discussion In all fat depots tested, GH-R mRNAs were detected. Similar transcripts were found in isolated adipocytes and



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Hours after last bGH injection FIG. 6. The effect of prolonged treatment with bGH on GH-R mRNA. Male rats were hx at 50 days of age and then given replacement therapy with L-T4 and hydrocortisone. One week after hypophysectomy, bGH (1 or 5 mg/kg-day) or saline was injected in two daily sc injections, at 0800 and 2000 h, for 6 days. The rats were killed 2, 6, or 12 h after the last injection. Sham-operated (Sham) and hx rats served as controls. GH-R mRNA levels were determined by a solution hybridization assay, as described in Materials and Methods. There were five rats in each group, individually analyzed. Vertical bars represent the SE. +, P < 0.01 vs. hx and 12 h; *, P < 0.01 vs. hx.

adipocyte precursor cells. At least 90% of the latter cell population has been shown to possess the ability to differentiate into adipocytes (21). Two transcripts (4.0 and 1.2 kb) were detected by a GH-R cRNA probe corresponding to the extracellular domain of the cloned rat hepatic GH-R. The long transcript corresponds in size to the rat hepatic GH-R transcript predicted to encode a 614-amino acid membraneanchored GH-R (8). The short transcript corresponds in size to the GH-BP transcript predicted to encode a 279amino acid soluble GH-BP in the rat (11). Both transcripts have been shown to be present in a variety of tissues, including liver, heart, kidney, skeletal muscle, skin, adrenal glands, intestine (12), and adipose tissue (13). Extracellular GH-BP has been shown to influence the tissue distribution of GH (22) and inhibit GH action in vitro (23). Immunoreactive GH-R/GH-BP, with the same estimated size as the GH-BP, has been detected in association with chromatin, suggesting a role for the GHBP in GH-regulated gene expression (24). A third, less abundant transcript (2.4-2.6 kb) has recently been demonstrated in rat adipose tissue (13) and rat liver (12). We found this low abundant transcript in both adipose tissue and isolated adipocytes of normal rats. At present it is not clear what these transcripts encode. With both Northern blot and solution hybridization assays we found decreased levels of GH-R mRNA after hypophysectomy, in line with previous reports (13). In contrast to previous observations, we found that GH treatment in doses within the physiological range resulted in an increase in GH-R mRNA levels. This discrepancy might be due to the experimental design, since

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we substituted the rats with T4 and hydrocortisone. Another possible explanation is differences in the assay reference (28S ribosomal RNA vs. actin). Induction of GH-R/BP mRNA levels by GH has recently been demonstrated in cultured chondrocytes (25). In spite of the fact that GH was given in a physiological dose, GH-R mRNA levels could not be restored. This could be an effect of low GH-R number when GH substitution started, since it has been shown that GH-R binding gradually declines after hypophysectomy (26). Another possibility is that loss of other pituitary factors, i.e. gonadotropins, is important in the regulation of GH-R mRNA. Earlier studies on GH-binding capacity to isolated adipocytes showed increased binding after GH treatment in vivo of hx rats (4, 6). GH binding has been shown to increase 3-4 h after GH injection of hx rats, with maximal binding after 6-8 h. Twelve to 16 h after the GH injection binding returned to the levels of hx control rats (4, 6). We found increased GH-R mRNA levels 2 h after the injection, and when 6 h had passed, a further increase was seen. Thereafter, GH-R mRNA levels declined. These results indicate that the changes in GH-R mRNA levels correlate well with changes in binding in adipocytes. Furthermore, the rapid regulation of the levels of GH-R transcripts is in line with the observation that there is a rapid decrease in GH-R mRNA after withdrawal of stimulation in cultured rat chondrocytes, with a half-life of 4 h (25). This rapid regulation of the GHR transcript is consistent with the presence of destabilizing sequences (ATTT) in the 3'-untranslated region of the rat GH-R cDNA (27). It must be pointed out that GH plasma levels are elevated for several hours after an iv injection of GH (28). The slow decline in GH-R mRNAs, in contrast to the rapid increase after a GH injection, seen in the present study, may, therefore, be due to sustained high levels of circulating GH. In normal male rats GH is secreted in episodic bursts, with low or undetectable levels between peaks (29, 30, 31). In view of this marked episodic secretion of GH, a possible relationship between secretion and tissue responsiveness may be regulated through these changes in receptors. Indeed, circumstantial evidence has indicated a relationship between GH plasma levels and the effects of the hormone in target tissues (32, 33). Few studies have addressed the question of whether the rapid turnover or hormonal regulation of GH-R has any implication for the diverse effects of the hormone. A correlation between binding and the insulin-like response to GH has been reported in adipocytes from normal and hx rats (5,14) and obese rats (34). The rapid effect of GH on its own receptor may indicate that its regulation is physiologically important. Goodman et al. (35) have recently demonstrated that temporally separable periods of RNA synthesis may be required for


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termination of the insulin-like effect of GH and the induction of refractoriness. GH has been shown to induce a marked increase in IGF-I mRNA in adipose tissue and isolated adipocytes, but this effect of GH appears later than the effect on the GH-R transcripts (Vikman, K., J. Isgaard, and S. Eden, unpublished observations). Obviously, GH induces several temporally separable changes at the level of gene expression in adipocytes. The present study has taken advantage of the cloned GH-R cDNA to study GH-R gene expression in adipocytes. However, the tools used do not permit evaluation of other possible GH-R proteins in adipocytes. Indeed, several studies have indicated that there may be differences in GH-R structure and function. For example, changes in the GH molecule may change its ability to induce the various effects. A noncovalently linked complex of residues 1-134 and 141-191 isolated from carbamidomethylated hGH has a lower potency to induce the insulin-like response to GH, whereas its ability to induce the lipolytic response is increased (36). Studies using the alkaloid swainsonine, which interferes with carbohydrate processing, and chemically modified forms of GH have indicated that a subclass of receptors mediates the lipolytic effect of GH (37). In addition, 20K and 22K GH have differences in affinity to the GH-BP and to GH-R (38). In studies using cross-linking techniques, several different bands of GH-R are usually detected (39-42). Furthermore, Godowski et al. (43) concluded, based on the cloned GH-R cDNA and the GH-R gene, that alternative splicing occurs in the 5'-coding region of the GHR mRNA. Whether such mechanisms are involved in expression of the different effects of GH is unclear. However, the high levels of GH-R expression and the well characterized effects of GH in adipocytes may provide a system to study alternatively processed GH-R and possible interactions between the GH-R and other membrane proteins in relation to the effect of GH.

Acknowledgments We thank Lawrence Mathews for the rat GH-R cDNA. We are grateful to Harriet Thelander and Ann-Sofie Lidmer for technical assistance.

References 1. Fagin KD, Lackey SL, Reagan C, DiGirolamo M 1980 Specific binding of growth hormone by rat adipocytes. Endocrinology 107:608-615 2. DiGirolamo M, Eden S, Engberg G, Isaksson 0, Lonnroth P, Hall K, Smith U 1986 Specific binding of growth hormone but not insulin-like growth factors by human adipocytes. FEBS Lett 205:15-19 3. Eden S, Schwartz J, Kostyo J 1982 Effects of preincubation on the ability of rat adipocytes to bind and respond to growth hormone. Endocrinology 111:1505-1512 4. Grichting G, Goodman H 1986 Growth hormone maintains its own receptors in rat adipocytes. Endocrinology 119:847-854

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5. Gause I, Eden S 1985 Hormonal regulation of growth hormone binding and responsiveness in adipose tissue and adipocytes of hypophysectomized rats. J Endocrinol 105:331-337 6. Gause I, Eden S 1986 Induction of growth hormone (GH) receptors in adipocytes of hypophysectomized rats by GH. Endocrinology 118:119-124 7. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood Wi 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537-543 8. Mathews LS, Engberg B, Norstedt G 1989 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905-9910 9. Trivedi B, Daughaday WH 1988 Release of GH binding protein from IM9 lymphocyte by endopeptidaes is dependent on sulphydryl group inactivation. Endocrinology 123:2201-2206 10. Smith WC, Kuniyoshi J, Talamantes F 1989 Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol 3:984-990 11. Baumbach W, Horner D, Logan J 1989 The rat growth hormonebinding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor. Genes Dev 3:1199-1205 12. Carlsson B, Billig H, Rymo L, Isaksson O 1990 Expression of the growth hormone-binding protein messenger RNA in the liver and extrahepatic tissues in the rat: co-expression with the growth hormone receptor. Mol Cell Endocrinol 73:R1-R6 13. Frick GP, Leonard JL, Goodman H 1990 Effect of hypophysectomy on growth hormone receptor gene expression in rat tissues. Endocrinology 126:3076-3082 14. Gause I, Isaksson O, DiGirolamo M, Smith U, Eden S1985 Changes in growth hormone binding and metabolic effects of growth hormone in rat adipocytes following hypophysectomy. Acta Physiol Scand 124:229-238 15. Bjorntorp P, Karlsson M, Pertoft H, Petterson P, Sjdstrom L, Smith U 1978 Isolation and characterization of cells from rat adipose tissue developing into adipocytes. J Lipid Res 19:316-324 16. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 17. de Leeuw WJF, Slagboom PE, Vijg J 1989 Quantitative comparison of mRNA levels in mamalian tissues: 28S ribosomal RNA level as an accurate internal control. Nucleic Acids Res 17:10137-10138 18. Durnam DM, Palmiter RD 1983 A practical approach for quantitating specific mRNAs by solution hybridization. Anal Biochem 131:385-393 19. Labarca C, Paigen P 1980 A simple, rapid and sensitive DNA assay procedure. Anal Biochem 102:344-352 20. Woolf CM 1968 Principles in Biometry. D van Nostrand Co., Princeton 21. Deslex S, Negrel R, Ailhaud G 1987 Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells. Exp Cell Res 168:15-30 22. Baumann G, Amburn KD, Buchanan TA 1987 The effect of circulating growth hormone-binding protein on metabolic clearance, distribution, and degradation of human growth hormone. J Clin Endocrinol Metab 64:657-660 23. Lim L, Spencer SA, McKay P, Waters MJ 1990 Regulation of growth hormone (GH) bioactivity by a recombinant human GHbinding protein. Endocrinology 127:1287-1291 24. Lobie PE, Barnard R, Waters MJ, The nuclear growth hormone receptor/binding protein. 72nd Annual Meeting of The Endocrine Society, Atlanta GA, 1990 25. Nilsson A, Carlsson B, Mathews L, Isaksson O 1990 Growth hormone regulation of growth hormone receptor mRNA in cultured rat epiphyseal chondrocytes. Mol Cell Endocrinol 70:237-246 26. Gause I, Eden S, Isaksson O, DiGirolamo M, Smith U1985 Changes in growth hormone binding and metabolic effects of growth hormone in rat adipocytes following hypophysectomy. Acta Physiol Scand 124:229-238 27. Shaw G, Kamen R 1986 A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659-667 28. Moore JA, Rudman CG, MacLachlan NJ, Fuller GB, Burnett B,



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Fane JW 1988 Equivalent potency and pharmacokinetics of recombinant human growth hormone with or without an N-terminal methionine. Endocrinology 122:2920-2966 Tannenbaum GS, Martin JB 1976 Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98:562-570 Eden S 1979 Age- and sex-related differences in episodic growth hormone secretion in the rat. Endocrinology 105:555-560 Jansson J, Eden S, Isaksson OP 1985 Sexual dimorphism in the control of growth hormone secretion. Endocr Rev 6:128-150 Isaksson O, Nutting DF, Kostyo J, Reagan CR 1978 Hourly variations in plasma concentrations of growth hormone and insulin and in amino acid uptake and incorporation into protein in diaphragm muscle of the rat. Endocrinology 102:1420-1428 Lindahl A, Eden S, Albertsson-Wikland K, Isaksson O, Kostyo J 1983 Relationship between the biological and immunological activities of growth hormone circulating in normal rats. Endocrinology 112:2054-2058 Landron D, Guerre-Millo M, Postel-Vinay MC, Lavau M 1989 Relationship between increased binding and insulin-like effect of human growth hormone in adipocytes from young fa/fa rats. Endocrinology 124:2305-2313 Goodman H, Tai L, Chipkin S 1990 The isoquinoline sulfonamide inhibitors of protein phosphorylation, H-7, H-8 and HA-1004, also inhibit RNA synthesis: studies on responses of adipose tissue to growth hormone. Endocrinology 126:441-450 Goodman H, Kostyo J 1981 Altered profiles of biological activity

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of growth hormone fragments on adipocyte metabolism. Endocrinology 108:553-558 Chipkin S, Szecowka J, Tai L, Kostyo J, Goodman H 1989 Different growth hormone-receptor interactions mediate insulin-like and lipolytic responses of rat adipose tissue. Endocrinology 125:450458 McCarter J, Shaw MA, Winer LA, Baumann G 1990 The 20,000 Da variant of human growth hormone does not bind to growth hormone receptors in human liver. Mol Cell Endocrinol 73:11-14 Moldrup A, Billestrup N, Thorn NA, Lernmark A, Nielsen JH 1989 Multiple growth hormone-binding proteins are expressed on insulin producing cells. Mol Endocrinol 3:1173-1182 Husman B, Haldorsen L, Andersson G, Gustafsson J 1988 Characterization of the somatogenic receptor in rat liver. J Biol Chem 263:3963-3970 Hocquette J-F, Postel-Vinay M-C, Djiane J, Tar A, Kelly PA 1990 Human liver growth hormone receptor and plasma binding protein: characterization and partial purification. Endocrinology 127:16651672 Smith WC, Talamantes F 1987 Identification and characterization of a heterogeous population of growth hormone receptors in mouse lever membranes. J Biol Chem 262:2213-2219 Godowski PJ, Leung DW, Meacham LR, Galgani JP, Hellmiss R, Keret R, Rotwein PS, Parks JS, Laron Z, Wood W 1989 Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Larontype dwarfism. Proc Natl Acad Sci USA 86:8083-8087


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