Regulation
of Sex Hormone-Binding Stephen
Globulin
Production
by Growth Factors
R. Plymate, Rita C. Hoop, Robert E. Jones, and Louis A. Matej
Sex hormone-binding globulin (SHBG) is a glycoprotein whose production has been demonstrated to be regulated by both sex steroids, as well as by thyroid hormone and peptide hormones such as insulin. However, none of these regulatory factors would explain the marked decrease in serum SHBG seen throughout the prepubertal and pubertal time period in both boys and girls. Furthermore, current in vitro data show that both androgens and estrogens can stimulate SHBG production by the human hepatoblastoma cell line Hep 62; yet, in vivo androgens appear to suppress SHBG levels, while estrogens are associated with elevated levels. This study was undertaken to determine possible mechanisms to explain this phenomenon. Hep G2 cell cultures were incubated with insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), or dehydroepiandrosterone (DHEA). Significant decreases in the level of SHBG in the culture medium relative to control cultures occurred for each of the growth factors (P < .O’l), whereas an increase in SHBG levels was observed in the medium of DHEA-treated cells. When cells were coincubated with IGF-I and thyroxine IT,). which alone stimulates SHBG production both in vivo and in vitro, the SHBG response to T, was blunted. These results suggest that growth factors, as well as insulin, may be important determinants in SHBG production. This is a US government work. There are no restrictions on its use.
U
NDERSTANDING the regulation of sex hormonebinding globulin (SHBG) levels in human plasma is important because SHBG, along with albumin, determines the bioavailability of sex steroids to most tissues, as well as the metabolic clearance of the steroids.‘-5 Previous studies have demonstrated that the production of this glycoprotein by a human hepatoma cell line (Hep G2) may be regulated by steroid hormones, thyroxine (T,), and peptides such as insulin and prolactin.6‘8 These studies were consistent with clinical situations such as obesity, hyperthyroidism, and prolactin-secreting tumors where SHBG levels were altered from those seen in normal individuals.‘-‘* However, they still did not explain the marked decrease in SHBG that was seen in both sexes as they proceed from early childhood through puberty.‘3-‘6 The decrease in SHBG during this period may be significant in that it increases the tissue availability of the constant low levels of sex steroids present in these individuals. In turn, this increased availability of sex steroids may be important in increasing growth and growth hormone secretion or in maturing the gonadotrophin-releasing hormone (GnRH) pulse generator of the hypothalamus. In an attempt to better understand the regulation of SHBG production during this crucial time period, the present study used the Hep G2 cell line as an in vitro model for the regulation of SHBG production. Although this is a transformed cell line, studies to date have demonstrated responses to hormonal stimuli that are consistent with in vivo studies. Further, using a cDNA probe for SHBG, mRNA bands on northern blots prepared from Hep G2 cell poly(A) + RNA are analogous to those found in normal human liver (R.C. Hoop, K.M. Wiren and S.R. Plymate, unpublished data). In the present study, the influence of various steroid and peptide hormones known to increase in vivo before puberty on SHBG production by Hep G2 cells was determined. These hormones included dehydroepiandrosterone (DHEA) and insulin-like growth factor I (IGF-I), since both represent factors that increase in boys and girls well in advance of clinical signs of puberty, but at a time when SHBG levels are known to decline.‘3-‘5
Metabolism, Vol39, No 9 (September). 1990:pp 967-970
MATERIALS AND METHODS
Hep G2 cells were kindly provided by Dr Barbara Knowles (Wistar Institute, Philadelphia, PA). Cells were grown to monolayers of 70% to 80% confluency in 25-cm’ plastic tissue culture flasks (Costar, Cambridge, MA) in 92% air and 8% CO,. The culture medium was Dulbecco’s Minimum Essential Medium (Gibco, Grand Island, NY) supplemented with 4% glutamine, 10% fetal calf serum (FCS; G&co), 100 U/mL penicillin, 100 pg/L streptomycin, and 2.5 pg/mL amphotericin. Before adding any of the test substances and after the cells had reached the specified density, monolayers were washed with FCS-free medium. (The medium used to dilute the inoculants was also serum-free.) Complete details of the culture system have been previously described.8 For each log dose shown, results from a minimum of 12 separate flasks were averaged. Transforming growth factor alpha (TGF-ru), and epidermal growth factor (EGF) were obtained from Peninsula Laboratories (Belmont, CA), and IGF-I was obtained from Bachem Bioscience (Philadelphia, PA). Thyroid and peptide hormones were prepared in a FCS-free medium; sodium hydroxide was added to dissolve T,. All solutions were adjusted to pH 7.4 before being added to the cells. The effects of the growth factors added to the Hep G2 cells were determined by inoculating the appropriate hormones daily in the culture medium at theconcentrations indicated in Tables 1 and 2. At the end of 72 hours of incubation, the media were removed and kept frozen at -20°C until assayed. The cells were harvested with 0.25% trypsin and counted in a hemacytometer. SHBG was measured by a radioimmunometric assay (Farmos Diagnostica, Oulunsalo, Finland). The intraassay and interassay coefficients of variation for this assay in our laboratory are 3.5% and 5.6%, respectively, for male and female serum pools. As previously
From the Department of Clinical Investigation, Madigan Army Medical Center, Tacoma, WA. The opinions or assertions contained herein are the private views of the authors and are not to be construed as oficial or rejecting the views of the Department of the Army or the Department of Defense. Address reprint requests to Stephen R. Plymate, MD, Department of Clinical Investigation, Box 454, Madigan Army Medical Center, Tacoma, WA 98431-5454. This is a US government work. There are no restrictions on its use. 00260495/90/3909-0015S0.00/0
967
PLYMATE
968
Table 1. SHBG Production
by Hep 62 Cells on the Third Day of Culture With IGF-I, TGF-a.
Hwmone
Concentration
Addition
(mol/L)
Control IGF-I
EGF
TGF-a
DHEA
EGF, or DHEA-S (*SEMI
SHBG/nmol
Medium SHBG nmol/106
Cells
ET AL
Cells/Flask
Total Protein mg
x 106
0
184.7 + 39.7
36.1 t 2.2
1.53 k 0.44
1om9
54.2 + 4.7
11.0 i 0.9t
2.80
i 0.55
loml'
66.5 + 8.3t
29.2 +1.3*
3.04
-f 0.28’
lom=
85.7 t 15.3t
30.6 zi 1.7'
2.29
t 0.73
10 l5
144.0 + 22.8
30.6 + 2.8
1.42
+ 0.27
1om7
102.0 * 10.2t
21.2 k 1.3t
2.24
f 0.55
1om9
86.6 k 8.8t
20.2 k 0.7t
2.62
k 0.79
10 "
100.1 + 21.7t
16.7 k 1.4t
2.32
zk 0.42
10 9
115.5 + 25.0*
30.6 I 2.0'
2.27
2 0.44
10 "
103.3 f 12.6t
24.5 + 1.3t
2.31
‘- 0.39
1om'3
143.5 e 8.6
22.7 + 3.1.
1.98 k 0.37
10 6
381.3 k 92.3*
90.7 zk 10.4t
2.01
+ 0.44
10 8
262.1 + 53.1
63.9 f 12.7"
2.42
t 0.48
‘P < .05 vcontrol. tP < .Ol Ycontrol. described, albumin,
corticosteroid-binding globulin, transferrin, and thyroxine-binding globulin did not cross-react in this assay.‘,” The results were expressed as nanomoles of SHBG per 10” cells. Total protein in the supernatant was measured using a dye affinity assay (Bio-Rad Laboratories, Richmond, CA). Each experiment was performed with six to eight culture flasks at each hormone dose and all experiments were repeated at least twice. Statistics were performed using the Statview II program (Calabasas, CA). One-way ANOVA was used as a test of significance between the various Hep
G2 treatment groups. RESULTS
As can be seen in Table 1, IGF-I, EGF, and TGF-(r treatments all resulted in a significant reduction in SHBG production by the Hep G2 cells. Treatment with DHEA, on the other hand, resulted in either no change or a significant increase in SHBG production by the cells (Table 1). IGF-I also suppressed total protein synthesis at the higher doses. When the change in SHBG was normalized for the change in total protein (SHBG/total protein ratio), similar results were obtained (Tables 1 and 2). Since thyroid hormone (T,) has been demonstrated to stimulate SHBG production both in vivo and in vitro in the Hep G2 cell line, IGF-I and T, were added to the cell culture system simultaneously to examine the combined effect of stimulating and suppressing factors.‘.‘* It has been shown previously that insulin can partially inhibit Table 2. SHBG Production
the stimulatory effect of T, on SHBG production.“,” In the present set of experiments, it was demonstrated that IGF-I can also partially suppress the effect of T, on SHBG in the Hep G2 cell line (Table 2). DISCUSSION
has been previously demonstrated that exogenously added insulin will suppress SHBG production by Hep G2 cells.*,‘* This finding is consistent with the relationship between serum insulin and SHBG in a number of clinical studies.‘,8,‘9-” However, the levels of insulin that had been demonstrated to be necessary for lowering SHBG levels both in vivo and in vitro were significantly higher than any increase noted during childhood and puberty and, therefore, could not account for the decrease in SHBG that is seen in both boys and girls before and during puberty.‘3,24 Since IGF-I is increasing during the time that SHBG is decreasing before puberty, and since this factor may act through the insulin receptor or, conversely, insulin through the IGF-I receptor, it seemed prudent to determine if such a growth factor could account for the pubertal decrease in SHBG.23,” In the present study, it was demonstrated that several growth factors can suppress SHBG production in the human hepatoma cell line used. These findings would be consistent with clinical studies recently reported demonstrating a de-
by Hep 62 Cells After 3 Days of Incubation With IGF-I, T,, or IGF + T,
Hormone
Concentration
Medium SHBG
Addition
hllol/L)
nmol/ 106 Cells
tP < .Ol
vcontrol.
SP < .05 vT,lO
'mol/L.
SEM) Cells/Flask
Total Protein ma
x 106
2.84
+ 0.75
lom"
106 _t 17.9'
37.5 +-2.9"
3.10
+- 0.78
10~15
110 * 7.3.
42.1 + 1.5'
3.30
k 0.55
10 7
327 + 16.0t
88.1 * 1.9t
2.60
+ 0.44
IGF-I
“P < .05 Vcontrol.
(+
51.6 k 1.5
0
IGF-IandT,
SHBG/nmol
153 t 21.1
Control
T,
It
lO~$
162 + 23.1
54.2 t 1.7
2.40
k 0.32
lo-" + IO_'
254 + 7.3tS
85.7 + 2.6t$
2.89
+ 0.88
10~15+
lo-'
276 + 21.3t$
63.3 + 6.3t$
2.52
+ 0.69
lo-" + 1om9
242 k 27.5t$
95.7 + 1.2tt
2.24
+ 0.44
10 IS+ 10 9
196 + 24
75.7 t 7.2
2.59
k 0.92
REGULATION
OF SEX HORMONE-BINDING
GLOBULIN
969
crease in SHBG subsequent to growth hormone administration, as well as cross-sectional studies in children demonstrating a close inverse correlation between IGF-I levels and SHBG.26-2x Whether the growth factors exert their effect on SHBG by acting through their specific growth factor receptors or through the insulin receptor has not been directly addressed by this study. In addition to IGF-I and insulin, TGF-LU and EGF can act through the same receptor. The concentrations of growth factors that have been demonstrated to suppress SHBG production in the Hep G2 cell lines are close to physiologic levels of the hormones in vivo. This observation may be indicative of an action through the specific receptor. Although the increase in IGF-I measured during childhood and into puberty has been demonstrated to correlate inversely with SHBG levels during this same time period, increases in DHEA and its sulfated product, DHEA-sulfate (DHEA-S), have also been correlated significantly with the decrease in SHBG in boys and girlsI However, as demonstrated in the present study, DHEA causes an increase in SHBG levels in cell culture medium. A similar androgenic effect on SHBG production by the Hep G2 cell line has also been noted for testosterone and DHT.6,8 Whether this effect of DHEA was due specifically to DHEA or conversion to another androgen was not specifically addressed by this study. However, significant increases in testosterone in the media of the DHEA-treated cells were not observed. Therefore, based on current data, it would appear that the most likely hormonal candidate for causing the decrease in SHBG in children up to and through puberty is IGF-I. The negative relationship between SHBG and IGF-I before puberty has recently been noted by Holly et al.” In addition to the changes that occur during puberty, IGF-I may also be the mediator of the decrease in SHBG seen with prolactin, since IGF-I levels are elevated in patients with prolactin-secreting tumors.29 In addition to the effects on SHBG, Holly et al have shown a similar correlation in vivo between insulin and IGF-I for IGF binding protein Iz7 This in vivo correlation has also been shown to occur in the production of IGF binding protein I by Hep G2 cells. 3o These data further suggest that, at least for the effects of peptide hormones, the Hep G2 cell line responds in a similar fashion predicted in vivo by correlative data. The importance of IGF-I and other growth factors in regulating SHBG following puberty will need to be further defined, especially in light of the recent demonstration by Caro et al of a significant decrease in IGF-I receptors in both the human and rat liver following puberty.3’ Indeed, if
insulin is a direct regulator of SHBG production in the obese adult, then there may be some suggestion for direct action through the insulin receptor rather than the IGF-I receptor, as discussed earlier. In spite of the mounting evidence for nonsteroid hormones as important physiologic regulators of serum SHBG levels, there are still significant differences in serum SHBG concentrations between men and women following the administration of sex steroids. Ain et a13*have shown that estrogen can affect thyroxine-binding globulin levels by increasing those isoforms of the protein that have a greater degree of glycosylation and hence a longer serum half-life, because the primary degradation of glycoproteins is through hepatic asialoglycoprotein receptors.33.34 In a previous study,35 we have presented data that sex steroids may induce similar isoform changes in circulating SHBG. Therefore, it can be hypothesized that steroid hormones may regulate SHBG levels at several points in synthesis. One point might be at the level of transcription or translation to increase the rate of synthesis of SHBG message or protein, respectively, while another level of control might be co- or posttranslational to alter the carbohydrate structure of the mature glycoprotein. This latter modification would subsequently affect the steadystate serum level of the protein by determining its turnover rate. Such steroid-induced changes in carbohydrate structure need not be specifically directed at altering SHBG levels, since they also appear to function on thyroxinebinding globulin. Recently, Chilton et al have shown that estrogen can stimulate N-acetylgalactosaminyl transferase (the first enzyme responsible for O-linked oligosaccharide chain biosynthesis) eightfold and oligosaccharyl-transferase (the enzyme required for N-linked oligosaccharide biosynthesis) lo- to 15fold in the developing rabbit endocervix. Therefore, the changes noted in protein glycosylation most likely represent a general effect of sex steroids on glycosylation enzymes, rather than one specifically directed towards SHBG.36 In summary, this report expands previous work in which peptide hormones, especially insulin, were demonstrated to be important regulators of SHBG production in the human Hep G2 cell line. Specifically, the present work suggests a mechanism for the decrease in SHBG seen during the prepubertal and pubertal period of growth in both boys and girls.
ACKNOWLEDGMENT
The authors gratefully acknowledge Eugenia R. Hough expert secretarial assistance in preparing the manuscript.
for her
REFERENCES 1. Pardridge WM, Mietus LJ: Transport of steroid hormones through the rat blood-brain barrier. J Clin Invest 64145-154, 1979 2. Pardridge WM, Mietus LJ: Transport of protein-bound steroid hormones into liver in viva. Am J Physiol237:E367-372, 1979 3. Pardridge WM: Plasma protein-mediated transport of steroid and thyroid hormones. Am J Physiol252:E157-164, 1987 4. Garden GA, Hannan CJ, Spencer CA, et al: The effect of sex hormone binding globulin (SHBG) on the pituitary testicular axis in hyperthyroid men. Steroids 52:385-386, 1988
5. Plymate SR, Petra PH, Namkung PC, et al: Modulation of serum estradiol (E,) levels and the metabolic clearance rate (MCR) of E, by acute changes in serum sex hormone binding globulin (SHBG) concentrations. Program of the Annual Meeting of the Society for Gynecologic Investigation, Baltimore, MD, 1988 (abstr) 6. Lee IR, Dawson SA, Wetherall JD, et al: Sex hormone binding globulin secretion by human hepatocarcinoma cells is increased by both estrogens and androgens. J Clin Endocrinol Metab 64:825-831, 1987
970
7. Rosner W, Aden DP, Khan MS: Hormonal influences on the secretion of steroid-binding proteins by a human hepatoma-derived cell line. J Clin Endocrinol Metab 59:806-808, 1984 8. Plymate SR, Matej LA, Jones RE, et al: Inhibition of sex hormone-binding globulin production in the human hepatoma (Hep G2) cell line by insulin and prolactin. J Clin Endocrinol Metab 67:460-464, 1988 9. Vermeulen A, Ando S, Verdonck L: Prolactinomas, testosterone-binding globulin and androgen metabolism. J Clin Endocrinol Metab 54:409-412, 1984 10. Winters SJ, Troen P: Altered pulsatile secretion of luteinizing hormones in hypogonadal men with hyperprolactinaemia. Clin Endocrinol21:257-263, 1984 11. Plymate SR, Fariss BL, Bassett ML, et al: Obesity and its role in polycystic ovary syndrome. J Clin Endocrinol Metab 52:12461248,1981 12. Wang C, Nieschlag E, Plymate SR, et al: Salivary testosterone in men: further evidence of a direct correlation with free serum testosterone. J Clin Endocrinol Metab 53:1021-1024, 1981 13. Bartsch W, Horst HJ, Derwahl KM: Interrelationships between sex hormone-binding globulin and 17fl-estradiol, testosterone, 5adihydrotesterone. thyroxine, and triiodothyronine in prepubertal and pubertal girls. J Clin Endocrinol Metab 50:1053-1056, 1980 14. Maruyama Y, Aoki N, Suzuki Y, et al: Sex-steroid-binding plasma protein (SBP), testosterone, oestradiol and dehydroepiandrosterone (DHEA) in prepuberty and puberty. Acta Endocrinol 114:6067,1987 15. Hall K, Enberg G, Ritzen M, et al: Somatomedin A levels in serum from healthy children and from children with growth hormone deficiency or delayed puberty. Acta Endocrinologica 94: 155165, 1980 16. Cunningham SK, Loughlin T, Culliton M, et al: Plasma sex hormone binding globulin levels decrease during the second decade of life irrespective of pubertal status. J Clin Endocrinol Metab 58:915-918, 1984 17. Hammond CL, Robinson PA: Characterization of a monoclonal antibody to human sex hormone binding globulin. FEBS Lett 168:207-212, 1984 18. Singh A, Hamilton-Fairley D, Koistsnen R, et al: Effect of insulin-like growth factor-type I (IGF-I) and insulin on the secretion of sex hormone binding globulin and IGF-I binding protein (IBP-I) by human hepatoma cells. J Endocrinol 124:1-3, 1990 19. Peiris AN, Mueller RA, Struve MF, et al: Relationship of androgenic activity to splanchnic insulin metabolism and peripheral glucose utilization in premenopausal women. J Clin Endocrinol Metab 64:162-169, 1987 20. Haffner SM. Katz MS, Stern MP, et al: The relationship of sex hormones to hyperinsulinemia and hyperglycemia. Metabolism 37:683-688, 1988 21. Shoupe D, Lobo RA: The influence of androgens on insulin resistance. Fertil Steril41:385-388, 1984 22. Evans DJ, Murray R, Kissebah AH: Relationship between muscle insulin resistance, insulin-mediated glucose disposal and
PLYMATE
insulin binding: Effects of obesity and body fat topography. Invest 74: I5 15 1525, 1984
ET AL
J Clin
23. Smith CP, Archibald HR, Thomas JM, et al: Basal and stimulated insulin levels rise with advancing puberty. Clin Endocrinol 28:7-14, 1988 24. Burke CL. Webber LS. Srinivasan SR, et al: Fasting plasma glucose and insulin levels and their relationship to cardiovascular risk factors in children: Bogalusa heart study. Metabolism 35:441446, 1986 25. Nissley SP, Reahler MM: Somatomedin/insulin factor tissue receptor. Clin Endocrinol Metab 13:43-67,
like growth 1984
26. Belgorosky A. Martinez A, Domene H, et al: High serum sex hormone-binding globulin (SHBG) and low serum non-SHBGbound testosterone in boys with idiopathic hypopituitarism: Effect of recombinant human growth hormone treatment. J Clin Endocrinol Metab65:1107-1111,1987 27. Holly JMP, Smith CP, Dunger DB, et al: Relationship between the pubertal fall in sex hormone-binding globulin (SHBG) and insulin-like growth factor binding protein I (IBP-1). A synchronized approach to pubertal development? Clin Endocrinol 31:277284, 1989 28. Rudd BT, Rayner PHW, Thomas PH: Observations on the role of GH/IGF-1 and sex hormone binding globulin (SHBG) in the pubertal development of growth hormone deficient (GHD) children. Acta Endocrinol279: l64- 169, 1986 29. Clemmons DR, Underwood LE. Ridgeway EC, et al: Hyperprolactinemia is associated with increased immunoreactivesomatomedin-C in hypopituitarism. J Clin Endocrinol Metab 52:731-735. 1981 30. Bailey DW, Mohan S, Rutanen E, et al: Evidence that the production of insulin-like growth factor binding protein-1 is regulated in human Hep-G2 cell line. Clin Res 38:216A, 1990 (abstr) 31. Caro JF, Poulos J, Ittoop 0, et al: Insulin-like growth factor I binding in hepatocytes from human liver. human hepatoma. and normal, regenerating, and fetal rat liver. J Clin Invest 8 I :976-98 I, 1988 32. Ain KB, Mori Y, Refetoff S: Reduced clearance rate of thyroxine-binding globulin (TBG) with increased sialylation: A mechanism for estrogen induced elevation of serum TBG concentration. J Clin Endocrinol Metab 65:689-696, 1987 33. Van Hall EV, Vaitukaitus JL, Ross CT, et al: Effects of progressive desialylation on the rat of disappearance of immunoreactive HCG from plasma in rats, Endocrinology 89: 1 l- 15, 197 1 34. Ashwell G, Steer CJ: Hepatic recognition and catabolism serum glycoproteins. JAMA 246:2358-2364, 1981
of
35. Hoop RC. Jones RE: Regulation of SHBG concentration by peptide and steroid hormones. Presented at the 71st Annual Meeting of The Endocrine Society, Seattle, WA, June 21-24, 1989 36. Chilton BS, Kaplan HA. Lennarz WJ: Estrogen regulation of the central enzymes involved in 0- and N-linked glycoprotein assembly in the developing and the adult rabbit endocervix. Endocrinology 123:1237-1244, 1988
of Sex Hormone-Binding Stephen
Globulin
Production
by Growth Factors
R. Plymate, Rita C. Hoop, Robert E. Jones, and Louis A. Matej
Sex hormone-binding globulin (SHBG) is a glycoprotein whose production has been demonstrated to be regulated by both sex steroids, as well as by thyroid hormone and peptide hormones such as insulin. However, none of these regulatory factors would explain the marked decrease in serum SHBG seen throughout the prepubertal and pubertal time period in both boys and girls. Furthermore, current in vitro data show that both androgens and estrogens can stimulate SHBG production by the human hepatoblastoma cell line Hep 62; yet, in vivo androgens appear to suppress SHBG levels, while estrogens are associated with elevated levels. This study was undertaken to determine possible mechanisms to explain this phenomenon. Hep G2 cell cultures were incubated with insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), or dehydroepiandrosterone (DHEA). Significant decreases in the level of SHBG in the culture medium relative to control cultures occurred for each of the growth factors (P < .O’l), whereas an increase in SHBG levels was observed in the medium of DHEA-treated cells. When cells were coincubated with IGF-I and thyroxine IT,). which alone stimulates SHBG production both in vivo and in vitro, the SHBG response to T, was blunted. These results suggest that growth factors, as well as insulin, may be important determinants in SHBG production. This is a US government work. There are no restrictions on its use.
U
NDERSTANDING the regulation of sex hormonebinding globulin (SHBG) levels in human plasma is important because SHBG, along with albumin, determines the bioavailability of sex steroids to most tissues, as well as the metabolic clearance of the steroids.‘-5 Previous studies have demonstrated that the production of this glycoprotein by a human hepatoma cell line (Hep G2) may be regulated by steroid hormones, thyroxine (T,), and peptides such as insulin and prolactin.6‘8 These studies were consistent with clinical situations such as obesity, hyperthyroidism, and prolactin-secreting tumors where SHBG levels were altered from those seen in normal individuals.‘-‘* However, they still did not explain the marked decrease in SHBG that was seen in both sexes as they proceed from early childhood through puberty.‘3-‘6 The decrease in SHBG during this period may be significant in that it increases the tissue availability of the constant low levels of sex steroids present in these individuals. In turn, this increased availability of sex steroids may be important in increasing growth and growth hormone secretion or in maturing the gonadotrophin-releasing hormone (GnRH) pulse generator of the hypothalamus. In an attempt to better understand the regulation of SHBG production during this crucial time period, the present study used the Hep G2 cell line as an in vitro model for the regulation of SHBG production. Although this is a transformed cell line, studies to date have demonstrated responses to hormonal stimuli that are consistent with in vivo studies. Further, using a cDNA probe for SHBG, mRNA bands on northern blots prepared from Hep G2 cell poly(A) + RNA are analogous to those found in normal human liver (R.C. Hoop, K.M. Wiren and S.R. Plymate, unpublished data). In the present study, the influence of various steroid and peptide hormones known to increase in vivo before puberty on SHBG production by Hep G2 cells was determined. These hormones included dehydroepiandrosterone (DHEA) and insulin-like growth factor I (IGF-I), since both represent factors that increase in boys and girls well in advance of clinical signs of puberty, but at a time when SHBG levels are known to decline.‘3-‘5
Metabolism, Vol39, No 9 (September). 1990:pp 967-970
MATERIALS AND METHODS
Hep G2 cells were kindly provided by Dr Barbara Knowles (Wistar Institute, Philadelphia, PA). Cells were grown to monolayers of 70% to 80% confluency in 25-cm’ plastic tissue culture flasks (Costar, Cambridge, MA) in 92% air and 8% CO,. The culture medium was Dulbecco’s Minimum Essential Medium (Gibco, Grand Island, NY) supplemented with 4% glutamine, 10% fetal calf serum (FCS; G&co), 100 U/mL penicillin, 100 pg/L streptomycin, and 2.5 pg/mL amphotericin. Before adding any of the test substances and after the cells had reached the specified density, monolayers were washed with FCS-free medium. (The medium used to dilute the inoculants was also serum-free.) Complete details of the culture system have been previously described.8 For each log dose shown, results from a minimum of 12 separate flasks were averaged. Transforming growth factor alpha (TGF-ru), and epidermal growth factor (EGF) were obtained from Peninsula Laboratories (Belmont, CA), and IGF-I was obtained from Bachem Bioscience (Philadelphia, PA). Thyroid and peptide hormones were prepared in a FCS-free medium; sodium hydroxide was added to dissolve T,. All solutions were adjusted to pH 7.4 before being added to the cells. The effects of the growth factors added to the Hep G2 cells were determined by inoculating the appropriate hormones daily in the culture medium at theconcentrations indicated in Tables 1 and 2. At the end of 72 hours of incubation, the media were removed and kept frozen at -20°C until assayed. The cells were harvested with 0.25% trypsin and counted in a hemacytometer. SHBG was measured by a radioimmunometric assay (Farmos Diagnostica, Oulunsalo, Finland). The intraassay and interassay coefficients of variation for this assay in our laboratory are 3.5% and 5.6%, respectively, for male and female serum pools. As previously
From the Department of Clinical Investigation, Madigan Army Medical Center, Tacoma, WA. The opinions or assertions contained herein are the private views of the authors and are not to be construed as oficial or rejecting the views of the Department of the Army or the Department of Defense. Address reprint requests to Stephen R. Plymate, MD, Department of Clinical Investigation, Box 454, Madigan Army Medical Center, Tacoma, WA 98431-5454. This is a US government work. There are no restrictions on its use. 00260495/90/3909-0015S0.00/0
967
PLYMATE
968
Table 1. SHBG Production
by Hep 62 Cells on the Third Day of Culture With IGF-I, TGF-a.
Hwmone
Concentration
Addition
(mol/L)
Control IGF-I
EGF
TGF-a
DHEA
EGF, or DHEA-S (*SEMI
SHBG/nmol
Medium SHBG nmol/106
Cells
ET AL
Cells/Flask
Total Protein mg
x 106
0
184.7 + 39.7
36.1 t 2.2
1.53 k 0.44
1om9
54.2 + 4.7
11.0 i 0.9t
2.80
i 0.55
loml'
66.5 + 8.3t
29.2 +1.3*
3.04
-f 0.28’
lom=
85.7 t 15.3t
30.6 zi 1.7'
2.29
t 0.73
10 l5
144.0 + 22.8
30.6 + 2.8
1.42
+ 0.27
1om7
102.0 * 10.2t
21.2 k 1.3t
2.24
f 0.55
1om9
86.6 k 8.8t
20.2 k 0.7t
2.62
k 0.79
10 "
100.1 + 21.7t
16.7 k 1.4t
2.32
zk 0.42
10 9
115.5 + 25.0*
30.6 I 2.0'
2.27
2 0.44
10 "
103.3 f 12.6t
24.5 + 1.3t
2.31
‘- 0.39
1om'3
143.5 e 8.6
22.7 + 3.1.
1.98 k 0.37
10 6
381.3 k 92.3*
90.7 zk 10.4t
2.01
+ 0.44
10 8
262.1 + 53.1
63.9 f 12.7"
2.42
t 0.48
‘P < .05 vcontrol. tP < .Ol Ycontrol. described, albumin,
corticosteroid-binding globulin, transferrin, and thyroxine-binding globulin did not cross-react in this assay.‘,” The results were expressed as nanomoles of SHBG per 10” cells. Total protein in the supernatant was measured using a dye affinity assay (Bio-Rad Laboratories, Richmond, CA). Each experiment was performed with six to eight culture flasks at each hormone dose and all experiments were repeated at least twice. Statistics were performed using the Statview II program (Calabasas, CA). One-way ANOVA was used as a test of significance between the various Hep
G2 treatment groups. RESULTS
As can be seen in Table 1, IGF-I, EGF, and TGF-(r treatments all resulted in a significant reduction in SHBG production by the Hep G2 cells. Treatment with DHEA, on the other hand, resulted in either no change or a significant increase in SHBG production by the cells (Table 1). IGF-I also suppressed total protein synthesis at the higher doses. When the change in SHBG was normalized for the change in total protein (SHBG/total protein ratio), similar results were obtained (Tables 1 and 2). Since thyroid hormone (T,) has been demonstrated to stimulate SHBG production both in vivo and in vitro in the Hep G2 cell line, IGF-I and T, were added to the cell culture system simultaneously to examine the combined effect of stimulating and suppressing factors.‘.‘* It has been shown previously that insulin can partially inhibit Table 2. SHBG Production
the stimulatory effect of T, on SHBG production.“,” In the present set of experiments, it was demonstrated that IGF-I can also partially suppress the effect of T, on SHBG in the Hep G2 cell line (Table 2). DISCUSSION
has been previously demonstrated that exogenously added insulin will suppress SHBG production by Hep G2 cells.*,‘* This finding is consistent with the relationship between serum insulin and SHBG in a number of clinical studies.‘,8,‘9-” However, the levels of insulin that had been demonstrated to be necessary for lowering SHBG levels both in vivo and in vitro were significantly higher than any increase noted during childhood and puberty and, therefore, could not account for the decrease in SHBG that is seen in both boys and girls before and during puberty.‘3,24 Since IGF-I is increasing during the time that SHBG is decreasing before puberty, and since this factor may act through the insulin receptor or, conversely, insulin through the IGF-I receptor, it seemed prudent to determine if such a growth factor could account for the pubertal decrease in SHBG.23,” In the present study, it was demonstrated that several growth factors can suppress SHBG production in the human hepatoma cell line used. These findings would be consistent with clinical studies recently reported demonstrating a de-
by Hep 62 Cells After 3 Days of Incubation With IGF-I, T,, or IGF + T,
Hormone
Concentration
Medium SHBG
Addition
hllol/L)
nmol/ 106 Cells
tP < .Ol
vcontrol.
SP < .05 vT,lO
'mol/L.
SEM) Cells/Flask
Total Protein ma
x 106
2.84
+ 0.75
lom"
106 _t 17.9'
37.5 +-2.9"
3.10
+- 0.78
10~15
110 * 7.3.
42.1 + 1.5'
3.30
k 0.55
10 7
327 + 16.0t
88.1 * 1.9t
2.60
+ 0.44
IGF-I
“P < .05 Vcontrol.
(+
51.6 k 1.5
0
IGF-IandT,
SHBG/nmol
153 t 21.1
Control
T,
It
lO~$
162 + 23.1
54.2 t 1.7
2.40
k 0.32
lo-" + IO_'
254 + 7.3tS
85.7 + 2.6t$
2.89
+ 0.88
10~15+
lo-'
276 + 21.3t$
63.3 + 6.3t$
2.52
+ 0.69
lo-" + 1om9
242 k 27.5t$
95.7 + 1.2tt
2.24
+ 0.44
10 IS+ 10 9
196 + 24
75.7 t 7.2
2.59
k 0.92
REGULATION
OF SEX HORMONE-BINDING
GLOBULIN
969
crease in SHBG subsequent to growth hormone administration, as well as cross-sectional studies in children demonstrating a close inverse correlation between IGF-I levels and SHBG.26-2x Whether the growth factors exert their effect on SHBG by acting through their specific growth factor receptors or through the insulin receptor has not been directly addressed by this study. In addition to IGF-I and insulin, TGF-LU and EGF can act through the same receptor. The concentrations of growth factors that have been demonstrated to suppress SHBG production in the Hep G2 cell lines are close to physiologic levels of the hormones in vivo. This observation may be indicative of an action through the specific receptor. Although the increase in IGF-I measured during childhood and into puberty has been demonstrated to correlate inversely with SHBG levels during this same time period, increases in DHEA and its sulfated product, DHEA-sulfate (DHEA-S), have also been correlated significantly with the decrease in SHBG in boys and girlsI However, as demonstrated in the present study, DHEA causes an increase in SHBG levels in cell culture medium. A similar androgenic effect on SHBG production by the Hep G2 cell line has also been noted for testosterone and DHT.6,8 Whether this effect of DHEA was due specifically to DHEA or conversion to another androgen was not specifically addressed by this study. However, significant increases in testosterone in the media of the DHEA-treated cells were not observed. Therefore, based on current data, it would appear that the most likely hormonal candidate for causing the decrease in SHBG in children up to and through puberty is IGF-I. The negative relationship between SHBG and IGF-I before puberty has recently been noted by Holly et al.” In addition to the changes that occur during puberty, IGF-I may also be the mediator of the decrease in SHBG seen with prolactin, since IGF-I levels are elevated in patients with prolactin-secreting tumors.29 In addition to the effects on SHBG, Holly et al have shown a similar correlation in vivo between insulin and IGF-I for IGF binding protein Iz7 This in vivo correlation has also been shown to occur in the production of IGF binding protein I by Hep G2 cells. 3o These data further suggest that, at least for the effects of peptide hormones, the Hep G2 cell line responds in a similar fashion predicted in vivo by correlative data. The importance of IGF-I and other growth factors in regulating SHBG following puberty will need to be further defined, especially in light of the recent demonstration by Caro et al of a significant decrease in IGF-I receptors in both the human and rat liver following puberty.3’ Indeed, if
insulin is a direct regulator of SHBG production in the obese adult, then there may be some suggestion for direct action through the insulin receptor rather than the IGF-I receptor, as discussed earlier. In spite of the mounting evidence for nonsteroid hormones as important physiologic regulators of serum SHBG levels, there are still significant differences in serum SHBG concentrations between men and women following the administration of sex steroids. Ain et a13*have shown that estrogen can affect thyroxine-binding globulin levels by increasing those isoforms of the protein that have a greater degree of glycosylation and hence a longer serum half-life, because the primary degradation of glycoproteins is through hepatic asialoglycoprotein receptors.33.34 In a previous study,35 we have presented data that sex steroids may induce similar isoform changes in circulating SHBG. Therefore, it can be hypothesized that steroid hormones may regulate SHBG levels at several points in synthesis. One point might be at the level of transcription or translation to increase the rate of synthesis of SHBG message or protein, respectively, while another level of control might be co- or posttranslational to alter the carbohydrate structure of the mature glycoprotein. This latter modification would subsequently affect the steadystate serum level of the protein by determining its turnover rate. Such steroid-induced changes in carbohydrate structure need not be specifically directed at altering SHBG levels, since they also appear to function on thyroxinebinding globulin. Recently, Chilton et al have shown that estrogen can stimulate N-acetylgalactosaminyl transferase (the first enzyme responsible for O-linked oligosaccharide chain biosynthesis) eightfold and oligosaccharyl-transferase (the enzyme required for N-linked oligosaccharide biosynthesis) lo- to 15fold in the developing rabbit endocervix. Therefore, the changes noted in protein glycosylation most likely represent a general effect of sex steroids on glycosylation enzymes, rather than one specifically directed towards SHBG.36 In summary, this report expands previous work in which peptide hormones, especially insulin, were demonstrated to be important regulators of SHBG production in the human Hep G2 cell line. Specifically, the present work suggests a mechanism for the decrease in SHBG seen during the prepubertal and pubertal period of growth in both boys and girls.
ACKNOWLEDGMENT
The authors gratefully acknowledge Eugenia R. Hough expert secretarial assistance in preparing the manuscript.
for her
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