Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450

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1 Pro. Natl. Aad. Si. USA Vol. 88, pp , August 1991 Biohemistry Interpulse interval in irulating growth hormone patterns regulates sexually dimorphi expression of hepati ytohrome P45 (liver gene expression/pulsatile pituitary hormone seretion) DAVID J. WAXMAN*t, NISAR A. PAMPORIt, PRABHA A. RAM*, ARUN K. AGRAWAL, AND BRNARD H. SHAPIRO: *Department of Biologial Chemistry and Moleular Pharmaology and Dana-Farber Caner Institute, Harvard Medial Shool, Boston, MA 2115; and tlaboratories of Biohemistry, University of Pennsylvania Shool of Veterinary Mediine, Philadelphia, PA 1914 Communiated by Paul Talalay, May 9, 1991 (reeivedfor review April 17, 1991) ABSTRACT Plasma growth hormone (GH) profiles are sexually differentiated in many speies and regulate the sexdependene of peripubesent growth rates and liver funtion, inluding steroid hydroxylase ytohrome P45 expression, by mehanisms that are poorly understood. By use of an external pump to deliver to hypophysetomized rats pulses of rat GH of varying frequeny and amplitude, a ritial element for liver disrimination between male and female GH patterns was identified. Liver expression of the male-speifi steroid 2a(orl6a)-hydroxylase P45, designated CYP2C11, was stimulated by GH at both physiologial and nonphysiologial pulse amplitudes, durations, and frequenies, provided that an interpulse interval of no detetable GH was maintained for at least 2.5 hr. This rmding suggests that hepatoytes undergo an obligatory reovery period after stimulation by a GH pulse. This period may be required to reset a GH-ativated intraellular signaling pathway or may relate to the short-term absene of GH reeptors at the hepatoyte surfae after a yle of GH binding and reeptor internalization. These requirements were distinguished from those neessary for the stimulation by GH of normal male growth rates in hypophysetomized rats, indiating that different GH responses and, perhaps, different GH-responsive tissues reognize distint signaling elements in the sexually dimorphi patterns of irulating GH. The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U.S.C solely to indiate this fat pisodi seretion is a general harateristi of many hormones, inluding pituitary-dependent hormones, panreati islet, and parathyroid hormones (1), and is often ruial to the triggering of hormone-dependent responses in target ells. For growth hormone (GH), seretion by the pituitary is not only intermittent, or pulsatile, but also the frequeny of pulsation is sex-dependent. In many speies, inluding the rat, hiken, and human (2-5), pituitary GH seretion is more frequent in females than in males. In the ase of the female rat, a high pulse frequeny results in GH present in irulation ontinuously, at levels ng/ml of plasma. This situation ontrasts with the intermittent presene of GH in plasma of male rats (2, 6). Studies in rats (7-1) and mie (11-13) have demonstrated that the expression of a number of sexually differentiated hepati proteins, inluding ytohrome P45 (P45)-linked steroid hydroxylases and drugmetabolizing enzymes, is primarily determined by plasma GH profiles and only seondarily regulated by the gonadal hormones through their effets on the hypothalamo-pituitary axis and its ontrol of GH seretion (14-16). The plasma GH profile in a male rat is haraterized by a pulse of 2-25 ng of GH per ml every 3-4 hr followed by a 2- to 2.5-hr period when irulating GH is nearly undetetable (.2 nrg/ml). It is unlear, however, just what features in this pattern are reognized as male by the hepatoyte. This ritial question is addressed by the present study, whih uses a reently developed external pump apparatus (17) for generation of periodi GH pulses of various frequeny and amplitude in hypophysetomized rats. Our findings lead us to onlude that neither the peak height, width, nor frequeny of irulating GH pulses is ritial, but rather a minimum length of trough period is key to the reognition of a GH pulse as masuline by adult rat hepatoytes. The signifiane of this observation is disussed in the ontext of possible mehanisms by whih GH seretory patterns regulate liver gene expression. MATRIALS AND MTHODS Animals. Adult intat and hypophysetomized male and female Sprague-Dawley rats were obtained from Charles River Breeding Laboratories. The animals were housed under onditions of regulated temperature (2-23C) and photoperiod (12 hr of light/12 hr of darkness; lights on at 8 hr) to minimize interanimal variation in GH seretory profiles, whih are light entrained (2). Rats were hypophysetomized by the supplier at the age of 8 weeks and were maintained for 4-5 weeks before doing the GH-replaement experiments. The effetiveness of hypophysetomy was verified by the absene of weight gain over this period. Hormonereplaement experiments with rat GH (rgh) (1.8 international units/mg; National Hormone and Pituitary Program) used three different protools: (i) ontinuous s.. infusion with an osmoti minipump (model 21, Alza) delivering 2 ng of rgh per g of body weight (BW) per hr for 7 days; (ii) s.. injetion at 12-hr intervals at ng of rgh per g of BW per injetion, as speified in the text; (iii) periodi injetion via a hroni indwelling right atrial atheter implanted and ontrolled by an external syringe pump, as desribed (17). For this last protool, injetions were applied as 3-min pulses at intervals ranging from 3 hr and 25 min to 12 hr and at doses of ng of rgh per g of BW per injetion, as speified in the text. ight-hour plasma GH profiles were determined by using an RIA with a sensitivity of 2-3 ng/ml (18). Repetitive blood sampling (.25 ml) at 15-min intervals was done on unrestrained, unstressed, and ompletely onsious rats outfitted with a mobile atheterization apparatus (19). Plasma GH profiles monitored on days 3 and 6 of the 7-day GH treatment period were indistinguishable. Abbreviations: GH, growth hormone; rgh, rat GH; BW, body weight; P45, ytohrome P45. tto whom orrespondene should be addressed at: Dana-Farber Caner Institute, Room JF-525, 44 Binney Street, Boston, MA 2115.

2 Biohemistry: Waxman et al. Pro. Natl. Aad. Si. USA 88 (1991) A MAL C HX + GH s. Cs-C 15 w O CL 75 ) I.-, C p1.1...i I... t. B FMAL 9: 11: 13: 15: 17: 9: 11: 13: 15: 17: Clok-time D HX + GH ont. f r-rrf-r, P45 Ativity and mrna Determinations. Liver mirosomes were isolated, and testosterone hydroxylase and 5a-redutase ativities were assayed, as desribed (1). P45 mrna levels were monitored by Northern (RNA) blotting using 3'P-labeled oligonuleotide probes speifi for individual P45s with methods desribed in detail elsewhere (2, 21). RSULTS As an initial approah to eluidating the ellular mehanisms that enable hepatoytes to disriminate between male and female GH seretary profiles, we undertook to identify whih of the salient features of a GH pulse-namely, its amplitude, duration, and/or interpulse interval-are the most ruial for stimulating the expression of a male-speifi P45 steroid 2a(orl6a)-hydroxylase, designated CYP2CI1 [aording to the reently adopted systemati nomenlature (22)]. ighthour plasma GH profiles of intat adult male and female rats are ompared (Fig. 1 A and B) with GH profiles determined for hypophysetomized adult male rats treated with rgh by using either an intermittent or a ontinuous hormone replaement regimen (Fig. 1 C and D). GH infusion using an osmoti minipump yielded a ontinuous presene of GH in plasma at levels omparable to those found in untreated adult female rats (Fig. 1D). This treatment stimulates expression of several female-predominant hepati enzymes (23-25). By on- 225 u-c 15 o $ CL 75 - I A 2P/DAY FIG. 1. Plasma GH profiles in adult male (A) and female rats (B) and in hypophysetomized (Hx) male rats treated with rgh by s.. injetion twie daily (C) or by ontinuous infusion (D). These plasma GH profiles are representative for individual intat rats and for hypophysetomized rats administered rgh for 7 days by s.. injetion, twie daily at 115 ng per g of BW per injetion, or by ontinuous s.. infusion with an osmoti minipump. trast, intermittent GH injetion (twie daily s..), ommonly used to mimi the pulsatile GH pattern present in untreated male rats, stimulates the expression of the male-speifi CYP 2C11 (refs. 7-1; also see below). Fig. 1C shows, however, that the resultant plasma GH profile is nonphysiologial, not only with respet to the frequeny of the pulse but also the duration of GH exposure, whih ontinued for at least 4 hr per injetion. To address the question of whether CYP 2C11 gene expression in intat male rats is stimulated by the natural GH pulses harateristi of this sex (e.g., Fig. 1A), we used a timerontrolled external syringe pump to generate regular, physiologial pulses of GH in hypophysetomized rats at intervals of 3 hr and 25 min, 4 hr, 6 hr, or 12 hr (i.e., seven, six, four, and two GH pulses per day). Analysis of the resultant plasma GH profiles in both male (Fig. 2) and female rats (data not shown) onfirmed the effetiveness of the external pump for deliven'ngreproduible amounts of GH at regular intervals. Physiologial plasma GH levels (peaks of 2-25 ng of GH per ml) were ahieved by administering a dose of 3-4 ng of rgh per g of BW per pulse; GH doses 2-fold lower yielded plasma GH peaks of 8-1 ng/ml, whereas 2-fold higher GH doses inreased peak hormone levels to the 4-6 ng/ml range, without notieable effet on pulse duration (data not shown). GH pulses administered in this manner were effetive in stimulating normal male growth rates (i.e., weight ) 1 a) a L- O P/DAY n{j "f - a^^nn I 'T...T I I : 11: 13: ~Va 15: 17: 9: 11: 13: Clok-time D 7P/DAY FIG. 2. Plasma GH profiles in hypophysetomized male rats administered GH by external pump. These GH profiles are representative for individual hypophysetomized male rats reeiving GH pulses (P) (3-32 ng of rgh per g of BW per injetion) two, four, six, or seven times daily for 7 days (A-D, respe- F- -Ttively). quivalent GH patterns were seen in 15: 17: GH-treated hypophysetomized female rats studied in parallel (data not shown).

3 687 Biohemistry: Waxman et al. Table 1. Influene of hypophysetomy and GH replaement on body weight (BW) gains BW gain, g per rat per week Male Female Intat* 19.6 ± ± 1.3t Hypox (-).4 ± 1.8t.3 ±.5t Hypox + atheterization (-)2.9 ± 2.2t (-)3.1 ± 2.1t Hypox + 7 P per day 18.2 ± ± 3.5 Hypox + 6 P per day 19.3 ± ± 3.8 Hypox + 4 P per day 16.8 ± ± 2.3 Hypox + 2 P per day 19.8 ± Hypox + 2 s.. per day ± 3.2 Hypox + ontinuous infusion 9.4 ± 2.2t 1. ± 2.1t The values are expressed as mean ± SD BW gain for at least 3 rats per group. Intat and hypophysetomized (Hypox) rats were untreated or were atheterized and then given two, four, six, or seven daily pulses (P) of rgh for 7 days at 3-4 ng per g of BW per pulse. Twie daily s.. injetions and ontinuous GH infusion were as desribed. *With or without atheterization. tp <.1 when ompared with intat male rats. All groups were signifiantly different from intat females at P <.1, exept for the Hypox + ontinuous infusion male and female groups. All groups were signifiantly different from the Hypox males and females (with or without atheterization) at P <.1. gain) in both male and female hypophysetomized rats, independent of whether the hormone was applied two, four, six, or seven times per day (Table 1). In ontrast, a lower, female-like growth rate was ahieved when GH was admin- 2. m 1. A MAL Q C UT HX s ) _ HX + GH, IIO C1 ~B FMAL a 2.- a) I-1. UT HX s HX + GH, FIG. 3. Influene of hypophysetomy (HX) and OH replaement on CYP2C11-dependent liver mirosomal testosterone 2ahydroxylase ativity. OH was administered to hypophysetomized male and female rats for 7 days by external pump at a frequeny of two, four, six, and seven pulses per day or by s.. (s) injetion twie per 24 hr, as indiated. Liver mirosomal ativities (mean ±SD) were determined for n = 3-4 male rats per group (A) or for n =2-4 female rats per group (B), and are both in units of nmol of 2ahydroxytetosteronefor"med perl mmir peir mg% ofmirsalpoen Indistinguishable profiles were obtained when CYP2C11-dependent testosterone 16a-hydroxylase ativity (15) was assayed. UT, untreated rats. A MAL Hx GH B UT 2C6- wwwwww o ) t CD - N o o Up ) Lo i 4) 4),- 4)4 - FMAL 2C11-2C11- Pro. Natl. Aad Si. USA 88 (1991) x - GH UT Hx s- S _-_-r.-' 2C6-3W"gg"g@ r_ I tso ui o 4)r N _ n- : 4) ) j.*u*t v4 ).! C4'I4 FIG. 4. Northern blot analysis of CYP2C11 mrna. Total liver RNA isolated from individual male (A) and female (B) rats was analyzed on Northern blots probed with 32P-labeled oligonuleotide probes speifi for CYP2C11 and for CYP2C6, whih served as a positive ontrol for RNA integrity and loading. Hypophysetomized rats (Hx) were treated with two to seven GH pulses per day, as indiated, and at the indiated GH doses (ng of rgh per g of BW per pulse or per s.. injetion). istered as a ontinuous infusion with an osmoti minipump (Table 1). The effets of these GH treatments on liver CYP2C11 expression were analyzed by monitoring CYP2C11-atalyzed liver mirosomal testosterone 2a-hydroxylation (Fig. 3). Several individual liver samples were also analyzed for CYP2C11 mrna levels (Fig. 4), whih in all ases paralleled the ativity values. As reported (7-1), CYP2C11 expression diminished greatly after hypophysetomy of male rats and was nearly undetetable in intat and hypophysetomized female rat liver. CYP2C11 expression in the hypophysetomized males was markedly stimulated by GH pulses delivered at the in vivo-like frequeny of six times daily, as well as by the less frequent, nonphysiologial frequenies of two and four times daily. Similar responses were obtained in hypophysetomized female rats, although the magnitude of the inrease was somewhat lower than that seen in the males (f. Figs. 3A and 3B). Comparable stimulatory responses were ahieved over a 7-fold range of GH doses per injetion and over a 3-fold range of total daily GH exposure in experiments done at two, four, and/or six GH pulses per day (Fig. 4 and data not shown). Surprisingly, however, GH pulsation at a frequeny of seven per day was ineffetive or only marginally effetive (Figs. 3 and 4) when tested in five independent experiments, despite the fat that both six and seven daily GH pulses stimulated normal male growth rates in the same groups of animals (Table 1). The dramati differene in the responsiveness of CYP2C11 to six vs. seven GH pulses per day (differing by only 35 min in interpulse interval) strongly suggests that to signal a male liver response, the minimal plasma GH trough time is highly restritive, and in the seven pulses per day hypophysetomized animals the trough may simply be too short to masulinize the liver with respet to CYP2C11 expression. Beause seven regular GH pulses per day were not reognized by the hepatoyte as masuline, we examined

4 whether this pulsation pattern presents to the liver signaling elements that are reognizable as feminine. Two responses that are onferred by a female (i.e., ontinuous) GH pattern were monitored: (i) indution of the female-predominant liver enzyme steroid 5a-redutase and the female-speifi CYP2C12 (23-25) and (ii) suppression of CYP3A2 and CYP2A2, adult male-speifi P45s distinguished from CYP2C11 by their apparent lak of dependene on GH pulsation for full expression (1). In these studies, seven daily GH pulses inreased steroid 5a-redutase expression in hypophysetomized male rat liver muh less effetively than did ontinuous GH infusion (Fig. SA). Moreover, seven daily GH pulses were unable to indue CYP2C12 mrna in the hypophysetomized rats, even though ontinuous GH treatment effeted a substantial elevation of this mrna (data not shown). GH pulsation at seven per day was also muh less effetive than ontinuous GH treatment with respet to suppression of CYP3A2 and -2A2 ativities and mrnas (Fig. 5B and data not shown). Thus, the full feminizing effets of GH on hepati gene expression are not imparted by the profile of seven daily GH pulses and are only realized when GH is present in irulation on a ontinuous basis. DISCUSSION GH seretory patterns are sexually differentiated in rats and other speies and onfer sex-dependent patterns of liver gene expression. These effets are partiularly striking in rodent liver (7-13), where they play a major role in the sexual differentiation of several important physiologial pathways and other proesses, inluding steroid hormone biotransformation, drug metabolism and pharmaokinetis, and hemial arinogen bioativation (14, 15, 28, 29). The ellular and moleular mehanisms by whih GH seretory patterns regulate liver gene expression are, however, largely unknown. The fat that in male rats the pituitary seretes GH in regular pulses separated by undetetable baselines, in ontrast to the more onstant GH seretion harateristi of females, has led to the reasonable, but unproven, hypothesis that the disrete pulses of GH are responsible for expression of the male phenotype (6, 14). In this study, we established that regular GH pulses generated in hypophysetomized rats markedly stimulate liver expression of the male-speifi CYP- C Biohemistry: Waxman et al. 2. :-_ 1. A 5aR B 6pHase UTHX 7 6 4ont UTHX ont HX+GH, HX+GH, FIG. 5. Inomplete feminization of hepati enzyme expression in hypophysetomized male rats by seven GH pulses per day. Feminization was monitored in isolated liver mirosomes by the elevation of the female-predominant testosterone 5a-redutase ativity (5aR) (A) and by the suppression of CYP3A2-dependent testosterone 6,8-hydroxylase (6(3Hase; male-speifi ativity) (B). Rat treatments and analysis of mirosomal enzyme ativities were as for Fig. 3. ont, ontinuous GH treatment, as in Fig. 1D. UT, HX, untreated and hypophysetomized male rat liver mirosomes. Ativities shown an be ompared to those of intat female liver mirosomes: and nmol/min per mg of protein for testosterone 5-redutase and 6,3-hydroxylase, respetively (n = 4). The inomplete feminization of these ativities in the ontinuous GH treatment group shown in this figure reflets the additional requirement of thyroxine for the full effet of GH (26, 27). Pro. Natl. Aad. Si. USA 88 (1991) C11. However, this masulinization of liver gene expression does not require a physiologial GH pulse amplitude or pulse duration. This fat is indiated by the effetiveness (Figs. 3 and 4) of both high and low peaks of GH generated by external pump, by the effetiveness of broad GH "pulses" generated by s.. GH injetions (Fig. 1C), and by our earlier finding that GH peak heights an be dereased by up to 9% in monosodium glutamate-treated rats without impairing the expression of CYP2C11 (18, 3). The effetiveness of lowamplitude GH pulses may be understood in terms of the high affinity of the GH reeptor for GH, Kd = 1-1 M (31), whih orresponds to half-maximal saturation of the reeptor at a plasma GH onentration of only 2 ng/ml. Masulinization of liver P45 expression also does not require the speifi GH pulse frequeny of six or seven per day that ours in intat, adult male rats (2, 6, 18) but, rather, appears dependent on a minimum interpulse trough interval. The present studies show that in the ase of a hypophysetomized rat model, the interpulse trough interval must be at least 2.5 hr in length. Aordingly, interpeak trough times of no detetable irulating GH (s2 ng/ml) that differ by as little as 35 min were found to give rise to distint patterns of liver gene expression. Interestingly, in the mouse, whih also expresses male-speifi and female-speifi P45s and other liver proteins under the influene of plasma GH patterns (11-13), GH seretion is pulsatile and ours at regular intervals in both males and females. In male mie, however, plasma GH pulses our eah 2.5 hr, giving rise to trough periods of undetetable GH almost 2 hr in length, whereas in females a shorter GH yling time of 1.4 hr results in trough times of <1 hr (32). Those findings support the present onlusion that a ritial minimum trough period, whih may vary from one speies to the next, is obligatory for liver expression of GH-regulated, male-speifi proteins, suh as CYP2C11. Our finding that hepatoytes require a minimum GH "off time" to express the masulinizing effets of pulsatile GH suggests that after stimulation by a GH pulse, male rat hepatoytes beome refratory to subsequent GH pulses until a minimum reovery period has elapsed. Persistent stimulation, suh as ours in female rats due to the ontinuous presene of GH in irulation, preludes suh a reovery, and leads to a distint-i.e.-feminine pattern of liver gene expression. The biohemial events underlying this obligatory reovery period are still undefined but may involve resetting of the intraellular signaling pathways triggered by GH (33-35) or, alternatively, may relate to the temporary unavailability of ell-surfae GH reeptors. In adult male rats, hepatoyte GH reeptors are apparently internalized after a plasma GH pulse and subsequently reappear at the ell surfae with an overall yling time of =3 hr (36, 37), a period that approximates the minimum interpulse interval seen in the present study. It is not known whether liver GH reeptor yling ours in the absene of ligand or whether it is diretly driven by the appearane of GH pulses at the ell surfae. Although the adaptation of GH-reeptor yling to GH pulses applied less frequently than in vivo (e.g., two or four GH pulses per day) would not pose a problem under a ligand-driven reeptor internalization model, hepatoytes may be unable to respond to GH pulses that reur more frequently than the endogenous male rhythm. Coneivably, in the seven-pulse-per-day experiments desribed in this report, the interpulse interval may be shorter than the minimum time needed for reappearane of unoupied GH reeptor at the ell surfae, a proess that probably requires new reeptor-protein synthesis (38). A reent analysis of liver P45 enzyme profiles in the dwarf rat strain NIMR/AS, whih is haraterized by a defet in GH synthesis that redues irulating GH by 9-95% (4), revealed essentially normal patterns of both female- and

5 6872 Biohemistry: Waxman et al. male-speifi P45 enzymes, inluding CYP2C11 (41), despite the great redution in plasma GH levels in this strain. This led Bullok et al. to propose that GH may not regulate the sexual differentiation of liver P45 expression (41). They further suggested that the apparent ations of GH indiated by earlier studies (7-12) may have, in part, resulted from nonsomatogeni properties of the bovine and human GH preparations used (41). This interpretation now appears unlikely beause in the present study rgh is shown to be fully effetive, both in masulinizing liver CYP2C11 expression when given in a suitable pulsatile manner (Figs. 3 and 4) and in feminizing hepati CYP2C12 and steroid 5a-redutase levels when given to intat male rats as a ontinuous infusion (data not shown). The possibility that NIMR/AS rats have low, but suffiient, levels of irulating GH [plasma peaks up to 15 ng/ml; ref. 4], as presented by Bullok et al. (41), seems the more likely explanation for their finding of normal P45 expression in these rats. Indeed, the somewhat elevated CYP2C11 levels in this strain, as well as the atual GH profiles desribed by Charlton et al. (4), are strikingly similar to those ahieved after indution of GH defiieny in adult rats by treatment with monosodium glutamate neonatally at a dose of 2 mg/g of BW, where near-normal liver P45 patterns are also observed (18, 3). Although low, GH levels in both models of GH defiieny are still signifiant when ompared to the Kd of the GH-GH reeptor omplex (31). The finding of normal P45 profiles in NIMR/AS rats is thus supportive of the present onlusion that it is the pattern of irulating GH, rather than the absolute magnitude of individual GH pulses, that is key to the sexual differentiation of liver phenotype. GH seretory profiles not only regulate hepati-gene expression but also play a ritial role in the peripubesent stimulation of somati growth, whih is more rapid in males under the influene of GH pulsation (6, 39). In our study, stimulation of normal male weight gain in hypophysetomized rats (a measure of somati growth) was ahieved in both males and females when physiologial GH pulses were given two, four, six, or seven times per day, whereas ontinuous GH exposure stimulated the lower growth rate harateristi of female rats. The effetiveness of seven GH pulses per day in restoring male growth but not male liver P45 expression leads us to onlude that these two GHstimulated responses have distint GH pulse requirements and that this may oneivably derive from a tissue-dependent reognition of signaling elements in the sexually dimorphi irulating GH patterns. 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