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Endocrinology Vol. 142, No. 11 4762-4770
Copyright © 2001 by The Endocrine Society

ARTICLES

Liver-Derived IGF-I Regulates GH Secretion at the Pituitary Level in Mice

Kristina Wallenius, Klara Sjögren, Xiao-Ding Peng, Seungjoon Park, Ville Wallenius, Jun-Li Liu, Mia Umaerus, Håkan Wennbo, Olle Isaksson, Lawrence Frohman, Rhonda Kineman, Claes Ohlsson and John-Olov Jansson

Research Centre for Endocrinology and Metabolism (K.W., K.S., V.W., O.I., C.O., J.-O.J.), Sahlgrenska University Hospital, Göteborg SE-413 45, Sweden; Department of Medicine (X.-D.P., S.P., L.F., R.K.), Section of Endocrinology and Metabolism, University of Illinois, Chicago, Illinois 60612; Department of Medicine (J.-L.L.), McGill University, Montréal QCH3A1A1, Canada; AstraZeneca R & D (M.U., H.W.), SE-43183 Mölndal, Sweden

Address all correspondence and requests for reprints to: John-Olov Jansson, Research Centre for Endocrinology and Metabolism, Gröna stråket 8, SE-413 45 Göteborg, Sweden. E-mail: john-olov.jansson{at}medic.gu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have reported that liver-specific deletion of IGF-I in mice (LI-IGF-I-/-) results in decreased circulating IGF-I and increased GH levels. In the present study, we determined how elimination of hepatic IGF-I modifies the hypothalamic-pituitary GH axis to enhance GH secretion. The pituitary mRNA levels of GH releasing factor (GHRF) receptor and GH secretagogue (GHS) receptor were increased in LI-IGF-I-/- mice, and in line with this, their GH response to ip injections of GHRF and GHS was increased. Expression of mRNA for pituitary somatostatin receptors, hypothalamic GHRF, somatostatin, and neuropeptide Y was not altered in LI-IGF-I-/- mice, whereas hypothalamic IGF-I expression was increased. Changes in hepatic expression of major urinary protein and the PRL receptor in male LI-IGF-I-/- mice indicated an altered GH release pattern most consistent with enhanced GH trough levels. Liver weight was enhanced in LI-IGF-I-/- mice of both genders. In conclusion, loss of liver-derived IGF-I enhances GH release by increasing expression of pituitary GHRF and GHS receptors. The enhanced GH release in turn affects several liver parameters, in line with the existence of a pituitary-liver axis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH SECRETION IN rodents is sexually dimorphic and pulsatile (1). Male rats have episodic bursts of GH secretion and low GH levels between the pulses, and female rats have higher basal interpulse GH levels and more frequent but lower amplitude pulses (2). Pituitary GH secretion is regulated by hypothalamic GH releasing factor (GHRF) and somatostatin. Increased hypothalamic GHRF secretion is followed by GH pulses, and somatostatin secretion increases during GH troughs (3, 4).

Several studies have shown that pharmacological treatment with high doses of IGF can inhibit GH secretion in both man and rodents (5, 6, 7, 8). The mechanisms mediating the inhibitory effect of IGF-I on GH secretion have been studied in GH-deficient animals treated with IGF-I or in vitro using pituitary cell cultures. There are, however, no studies on the mechanisms mediating IGF-I feedback in animals with intact GH secretion.

In primary pituitary cell cultures, IGF-I has been shown to suppress both basal and GHRF-stimulated GH release and synthesis. Thus, it has been suggested that IGF-I can inhibit the stimulatory effect of GHRF on GH release directly at the pituitary level (9, 10). In GH-deficient rodents, IGF-I treatment suppresses the increased GHRF receptor expression in these animals (11). On the other hand, central administration of IGF-I to GH-deficient rats decreases GHRF and increases somatostatin expression, suggesting that IGF-I can also act at the hypothalamic level (12). In line with this, it has been shown that IGF-I is locally produced in the hypothalamus (13).

IGF-I could also inhibit GH secretion by regulating the expression of the GH secretagogue receptor (GHS-R). Activation of the GHS-R with synthetic GH secretagogues (GHS), or the endogenous ligand Ghrelin, induces GH secretion (14, 15). GHS-R activation stimulates GH release directly at the pituitary level (16) and increases hypothalamic GHRF release and may inhibit hypothalamic somatostatin release (14, 17). It is unclear, however, whether endogenous GHS-R ligands contribute to the regulation of GH pulsatility.

The secretory pattern of GH regulates several sexually dimorphic liver functions in rodents, such as expression of major urinary protein (MUP) and the PRL receptor (PRL-R) (18, 19, 20, 21). MUP is expressed at about three times higher levels in livers of male, compared with female rodents, and PRL-Rs are expressed at higher levels in females (18). Furthermore, continuous treatment of male mice with GH leads to suppression of MUP and induction of PRL-R expression (18). Therefore, MUP and PRL-R expression are markers of GH trough levels.

Mice with liver-specific IGF-I knockout have 80% decreased serum IGF-I levels and increased circulating GH levels (22, 23). In the present study, we have investigated how elimination of hepatic IGF-I modifies the hypothalamic-pituitary GH axis to enhance GH secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Transgenic mice were bred and recombination was induced by interferon treatment at 4 wk of age, as described earlier (22). Interferon-treated siblings, homozygous for loxP but lacking Mx-Cre, were used as controls. The mice were killed by cervical dislocation at 2.5–3 months of age and organs were collected and weighed and then immediately snap frozen in liquid nitrogen. All experiments were conducted in accordance with institutional guidelines and were approved by the local committee for animal care.

RIA of IGF-I and GH
Plasma was obtained by centrifuging heparinized capillaries with blood obtained from the tip of the tail of unanesthetized mice at different times throughout the day. Plasma IGF-I levels were measured 3 wk after interferon treatment by a double-antibody IGF binding protein-blocked RIA according to Blum and Breier (24). Mouse GH levels were measured by RIA (RPA 551, purchased before November 1999; Amersham Pharmacia Biotech, Little Chalfont, UK), according to the manufacturer’s instructions, with a detection range of 1.3–100 ng/ml. Mouse GH was also measured (see Fig. 5BGo) as described previously (25) using reagents kindly supplied by NHPP, NIDDK, and Dr. Parlow.



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  		Figure 5. GH release after treatment of LI-IGF-I-/- mice and control mice with GHRF and GHS. A, Male mice treated with GHRF (40 µg/kg ip); B, female mice treated with GHRF (40 µg/kg ip); C, male mice treated with the GHS ipamorelin (500 µg/kg ip; pooled data from two experiments); and D, female mice treated with the GHS ipamorelin (500 µg/kg ip). Blood samples were taken before treatment and then after 15, 30, and 60 min. There were four to nine mice in each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. corresponding control mice.

 
Measurement of MUP
One microliter of urine from mice was run on 10% NuPage Bis-Tris gels in MES-buffer (Novex, San Diego, CA) (18). MUP, with a size of about 20 kDa, was the dominant protein on gels. Gels were Coomassie stained and MUP was quantified by scanning and densitometric analysis using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Because an overwhelming proportion of all protein in urine was MUP, measurement of total protein with the Dc protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA) based on the Lowry method was also used to quantitate MUP levels. BSA (fraction V) was used as a relative standard (Sigma, St. Louis, MO).

Real-time RT-PCR of IGF-I mRNA in hypothalamus and liver
First-strand cDNA was synthesized from 1 µg of total RNA from liver and hypothalamus using Superscript II RT (Life Technologies, Inc., St. Louis, MO) with random hexamers according to the manufacturer’s instructions. Taqman-PCR was performed with the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) using VIC-labeled fluorogenic probes specific for either the IGF-I transcript or the internal standard M36B4. Oligo primers and probes (Table 1Go) were chosen using the Primer Express software (Applied Biosystems). The PCR was performed using Taqman Universal PCR Mastermix (Applied Biosystems) to which primers and probes were added (final concentrations 400 nM and 200 nM, respectively). Each run included reactions for the specific gene, IGF-I, the internal standard, and negative controls for both primer sets. All samples were run in triplicate in 96-well plates in the ABI Prism 7700 sequence detector according to the manufacturer’s standard protocol. For both primer sets, serial dilutions were conducted with different cDNA preparations to confirm the kinetics of the PCR. These analyses verified that the efficiencies of amplification were equal for both primer sets and thereby allowing quantification by the comparative CT method (user bulletin #2, Applied Biosystems).


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  		Table 1. Primer sequences for Taqman RT-PCR for mouse IGF-I mRNA levels

 
Multiplex RT-PCR for pituitary GHRF-R, GHS-R, and somatostatin receptor (sst) 1–5 mRNA
The relative levels of pituitary receptor mRNA were measured by multiplex RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. One microgram of total RNA was reverse transcribed using the Superscript preamplification system for first-strand synthesis (Life Technologies, Inc.) with random hexamer priming in 20 µl volume. The resultant cDNA was used in two separate PCR mixtures containing specific primers for GHRF-R, GHS-R, and GAPDH (reaction #1) or sst1-5 and GAPDH (reaction #2). Primer sequences used in each reaction are shown in Table 2Go. Reaction conditions and validation procedures for the multiplex RT-PCR have been previously described (26). There was no significant difference in GAPDH mRNA levels between experimental groups. Therefore, signal intensity for each of the pituitary receptor subtypes was adjusted by that of GAPDH to control for variability in the amount of total RNA used in the reverse transcription reaction and the efficiency of conversion of RNA to cDNA.


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  		Table 2. Primer sequences, cycling number, and annealing temperature of multiplex RT-PCR for mouse pituitary receptor mRNA levels

 
Riboprobes
The mouse PRL-R cDNA probe identifies the intracellular PRL-R sequence (27). The plasmid was linearized using XhoI, and the antisense [32P]CTP-labeled PRL-R RNA probe was synthesized using T3 RNA polymerase. Mouse hypothalamic cDNA was amplified by PCR using primers for mouse GHRF, somatostatin, NPY, or ß-actin, and pituitary cDNA was amplified using primers for mouse GH to generate riboprobes. The primer sequences and probe sizes for GHRF, somatostatin, NPY, and ß-actin have been previously reported (26). Primer sequences used to generate riboprobes for mouse GH were sense: 5'-CTGGCTGCTGACACCTACAAA-3' and antisense: taatacgactcactatagggagagttcaagctggtccaCAGGAGAGCAGCCCATAGTTT (capital letters indicated mouse GH gene-specific sequences, GenBank Accession x02891 and K03232). The antisense primers were modified to contain a 17-base T7 RNA polymerase recognition sequence (5'-TAATACGACTCACTATA-3'), a 6-base transcription initiation sequence, and 15 or 20 bases of nonspecific sequence appended at the 5' end. The nonspecific sequence was added to the primer to allow for the differentiation of protected and unprotected probe following RNase digestion. The amplified PCR products were used as templates for in vitro transcription performed using the MAXIscript kit (Ambion, Inc., Austin, TX) in the presence of [{alpha}-32P]CTP. Radiolabeled riboprobes were gel purified before use.

Ribonuclease protection assays (RPAs)
Liver RNA for PRL-R was prepared from frozen liver according to Chomczynski and Sacchi (28). Mouse PRL-R mRNA levels in the liver were measured by the RPA II kit (Ambion, Inc.). The assay was performed according to the manufacturer’s instructions using 40-µg liver RNA per sample, with 18S as an internal standard (Ambion, Inc.). RPA for hypothalamic GHRF, somatostatin, and NPY mRNA and for pituitary GH mRNA was performed using HybSpeed RPA kit (Ambion, Inc.) following the manufacturer’s instructions with minor modifications. The riboprobes were mixed in two reactions: reaction #1: GHRF [2 x 104 cpm; specific activity, 1 x 109 cpm/µg], somatostatin [1 x 104 cpm; specific activity, 3 x 108 cpm/µg], NPY [2 x 104 cpm; specific activity, 9 x 108 cpm/µg], and ß-actin [4 x 103 cpm; specific activity, 8 x 107 cpm/µg; reaction #2: GH [5 x 103 cpm; specific activity, 4 x 107 cpm/µg] and ß-actin [1 x 104 cpm; specific activity, 3 x 108 cpm/µg]. The mixture was incubated for 20 min at 68 C in 10 µl of HybSpeed hybridization buffer containing 50% of the total RNA isolated from a single hypothalamus (reaction #1), 1 µg of mouse pituitary RNA (reaction #2) or 50 µg of yeast RNA (negative control). Unhybridized probes for all RPAs were digested by treating the reactions with RNase A/T1 mix (1.0 µg/20 U) for 1 h at 37 C. Protected fragments were separated by electrophoresis through a 5% polyacrylamide/8 M urea gel. Gels were dried on chromatography paper and exposed to a PhosphoImager screen. Band intensity was evaluated using a PhosphoImager and ImageQuant software (Molecular Dynamics, Inc.).

Treatment with GHRF and the GHS, ipamorelin
Mice were anesthetized with a mixture of ketamine and medetomidine just before the first blood sample was taken. They were then immediately injected ip with GHRF (40 µg/kg) or the GHS ipamorelin (500 µg/kg) (29). Blood samples were collected 15, 30, and 60 min after injection.

Statistical analysis
Differences between groups were compared by t test with the exception of circulating GH data, in which the {chi}2-test was used. Logarithmic transformation was used where appropriate. Values are given as means and SEM. P values of <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma IGF-1 and liver and hypothalamic IGF-I mRNA levels
Serum levels of IGF-I were decreased by 80%, and liver mRNA levels were decreased by 90% in both male and female liver-specific IGF-I knockout (LI-IGF-I-/-) mice, compared with control mice (Fig. 1Go, A and B). These data are in line with previous pooled data from male and female mice (22, 23). Hypothalamic IGF-I mRNA levels were increased by 31% in the female LI-IGF-I-/- mice (P < 0.01; Fig. 1CGo). There was a similar tendency in the male LI-IGF-I-/- mice, but this effect was not statistically significant (P = 0.05; Fig. 1CGo).



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  		Figure 1. IGF-I levels in serum and IGF-I mRNA in liver and hypothalamus in LI-IGF-I-/- mice, compared with corresponding control mice. A, IGF-I levels in serum were measured when the mice were 7 wk old by a RIA. IGF-I mRNA levels in liver (B) and hypothalamus (C) were measured in 2.5-month-old mice by the Taqman real-time PCR. There were five to six mice in each group. **, P < 0.01 vs. corresponding control mice.

 
Plasma GH, pituitary GH mRNA levels, and liver weight
Plasma GH levels were analyzed by a {chi}2 test dividing the data into two groups, above or below 1.3 ng/ml. A significantly greater proportion of GH values were above 1.3 ng/ml in male LI-IGF-I-/- mice, compared with control mice (53% vs. 24%, {chi}2 = 6.31, P < 0.02, n = 34–36), and in female LI-IGF-I-/- mice, compared with control mice (62% vs. 34%, {chi}2 = 8.15, P < 0.01, n = 47–59). These data are in line with earlier results obtained from pooled male and female data (22, 23). Pituitary GH mRNA levels did not differ between male (14.9 ± 1.0 vs. 14.8 ± 0.7 arbitrary densitometric units, n = 5) and female (15.2 ± 1.0 vs. 14.2 ± 1.1 arbitrary densitometric units, n = 5–6) LI-IGF-I-/- and control mice. As previously reported (22), liver-specific elimination of IGF-I did not significantly affect body weight at this age (data not shown). However, relative liver weight (percent liver weight/body weight) was significantly higher in both male and female LI-IGF-I-/- mice, compared with control mice (male: 6.4 ± 0.1% vs. 5.7 ± 0.1%, P < 0.01 and female: 7.0 ± 0.2% vs. 6.1 ± 0.1%, P < 0.01), as previously shown with pooled male and female data (22).

MUP and hepatic PRL-R mRNA levels
MUP levels were analyzed by gel electrophoresis and Coomassie staining of urine samples from LI-IGF-I-/- and control mice (Fig. 2AGo). Densitometric scanning of gels showed that the MUP levels were three times higher in urine from control males, compared with control females, confirming earlier results by Nordstedt and Palmiter (18). The MUP levels were decreased by 28% in male LI-IGF-I -/- mice, compared with control males (11.2 ± 1.0 vs. 15.7 ± 1.0 ODu*mm2, P < 0.02, n = 5–6). There was no difference in the MUP levels between female LI-IGF-I-/- mice and controls. MUP levels (reflected by total protein in urine) were also measured by the Lowry method and were markedly lower in male LI-IGF-I-/- mice, compared with male control mice (Fig. 2BGo).



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  		Figure 2. MUP in urine from male and female, LI-IGF-I-/- mice, compared with corresponding control mice. A, Representative gel showing urine analyzed by gel electrophoresis followed by Coomassie staining. B, The relative protein content in urine from male and female LI-IGF-I-/- and control mice was quantified by the Lowry method. There were 10–15 mice in each group. **, P < 0.01 vs. corresponding control mice.

 
Expression of PRL-R mRNA in the liver was measured in LI-IGF-I-/- mice and control mice using RPA (Fig. 3AGo). Densitometric scanning showed that the PRL-R mRNA levels were significantly higher in the livers of male LI-IGF-I-/- mice, compared with male control mice (Fig. 3BGo). There was no significant difference in hepatic PRL-R mRNA levels between female LI-IGF-I-/- and female control mice (Fig. 3BGo). Taken together, the results of Figs. 2Go and 3Go indicate that these GH-regulated hepatic functions are altered in male LI-IGF-I-/- mice in a manner consistent with these mice having increased GH trough levels (18).



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  		Figure 3. PRL-R mRNA levels in the liver of male and female LI-IGF-I-/- mice, compared with corresponding control mice. A, Two representative gel electrophoresis lanes from each experiment group. B, Results of densitometric quantification of two gels. There were four to six mice in each group. **, P < 0.01 vs. corresponding control mice.

 
Expression of hypothalamic neuropeptides and pituitary receptors that regulate GH secretion
To investigate how liver-derived IGF-I affects GH secretion, the expression of hypothalamic GHRF, somatostatin, and NPY and pituitary receptors for GHRF, GHS, and somatostatin was measured in LI-IGF-I-/- and control mice. GHRF, somatostatin, and NPY mRNA levels in the hypothalamus of LI-IGF-I-/- mice were not significantly altered (Fig. 4Go, A and B). In contrast, the expression of the receptors for GHRF and GHS were increased in both male and female LI-IGF-I-/- mice, compared with control mice (Fig. 4Go, C and D). GHRF-R levels were increased by 26% in male and by 70% in female LI-IGF-I-/- mice, and the GHS-R levels were increased by 74% in male and by 112% in female LI-IGF-I-/- mice. The mRNA levels of the different forms of the sst1–5 in the pituitary were not significantly affected in these mice (Figs. 4Go, E and F). These results demonstrate that circulating liver-derived IGF-I regulates GH release at the pituitary level rather than at the hypothalamic level.



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  		Figure 4. Expression of GHRF, somatostatin, and NPY in the hypothalamus and GHRF-R, GHS-R, and ssts in the pituitary of LI-IGF-I-/- male and female mice compared with control mice. GHRF, somatostatin, and NPY mRNA levels were measured by RPA (A, males; B, females). GHRF-R, GHS-R (C, males; D, females), and sst1–5 mRNA levels (E, males; F, females) were measured by multiplex RT-PCR. There were five to six mice in each group. *, P < 0.05 vs. corresponding control mice.

 
Response to GHRF and GHS
To determine whether the increase in expression of GHRF and GHS-Rs in LI-IGF-I-/- mice results in an enhanced sensitivity to GHRF and GHS, we compared the GH response to these substances. Male and female LI-IGF-I-/- mice showed a rapid and pronounced response to GHRF with peak levels of GH 15 min after treatment, but the control mice did not respond to this dose of GHRF (Fig. 5Go, A and B). Both male LI-IGF-I-/- and control mice responded to GHS treatment with peak levels of GH at 15 min after treatment (Fig. 5CGo). However, at 30 and 60 min after treatment, the GH levels were significantly higher in LI-IGF-I-/- mice, compared with control mice (Fig. 5CGo). Treatment of female LI-IGF-I-/- mice with GHS increased GH levels to nearly 800 ng/ml at 15 min, and GH remained elevated at 30 and 60 min, but this dose of GHS did not increase GH levels at all in the control females (Fig. 5DGo). These results demonstrate that the LI-IGF-I-/- mice had increased responsiveness to GHRF and GHS, which is consistent with their increased expression of the receptors for GHRF and GHS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By using a unique mouse model for inducible and liver-specific IGF-I depletion, we demonstrated that liver-derived IGF-I exerts a tonic inhibitory effect on GH-secretion in mice. GH levels were increased in these mice showing that GH cannot normalize its own secretion via short-loop feedback in the absence of liver-derived IGF-I. Investigation of a patient with complete depletion of IGF-I showed that both pulse height and the basal GH levels were increased (30). The increased GH levels in this patient could be reversed by IGF-I replacement (31). Therefore, it is clear that endogenous IGF-I suppresses GH secretion in humans as well as in mice. The present results extend those reported by Woods et al. (30) on the IGF-I-deficient patient by showing that GH secretion is modulated mainly by liver-derived IGF-I that constitutes 80% of circulating IGF-I (present results, 22, 23).

In humans and some other species, GH levels are increased during fasting (32, 33). A physiological implication of the negative feedback effect by IGF-I in humans has been demonstrated by Hartman et al. (32), who showed that the enhanced GH secretion in humans during fasting is caused by a decrease in circulating IGF-I levels, presumably owing to decreased hepatic IGF-I production. The increase in GH production could in turn be of importance for the lipolysis and insulin antagonism during fasting. This theory is supported by recent findings by us and others that the mice with liver-specific IGF-I depletion had decreased fat mass and decreased insulin sensitivity and that these effects may be mediated by the increased GH secretion (34, 35).

On the basis of earlier studies with exogenous IGF-I treatment, the negative feedback effect of IGF-I on GH secretion could be exerted either in the hypothalamus [e.g., via suppressed GHRF or enhanced somatostatin release (7, 8, 9, 36)], or directly at the pituitary level (9, 10, 36). Our data support the latter hypothesis. Reduction of circulating IGF-I by 80% increased GHRF-R and GHS-R mRNA levels in pituitaries from LI-IGF-I-/- mice. These data are consistent with the finding that GH receptor–null mice, which have a decrease in both direct effects of GH and serum IGF-I levels (37), also have increased GHRF-R and GHS-R mRNA levels (26). Sugihara et al. (38) demonstrated that IGF-I decreased GHRF-R mRNA levels in primary rat pituitary cell cultures. An inhibitory effect of IGF-I on GHRF-R mRNA levels has also been reported in vivo using IGF-I replacement in the GH-deficient spontaneous dwarf rat (11). In this same model, IGF-I treatment had no effect on pituitary GHS-R expression though GH treatment did suppress GHS-R expression (39). One possible explanation for our present data in conjunction with those of Kamegai et al. (39) is that IGF-I can suppress GHS-R expression in the presence, but not in the absence, of an IGF-I independent, direct GH action.

There was no effect of depletion of liver-derived IGF-I on expression of the hypothalamic neuropeptides GHRF, somatostatin, and NPY, all of which participate in regulation of GH release (1, 3, 4, 36, 40, 41). This is consistent with previous reports that systemic IGF-I treatment does not affect the expression of GHRF or somatostatin in GH-deficient rats (12). Hypothalamic IGF-I was significantly increased in female LI-IGF-I-/- mice with a similar tendency in males. This increase in hypothalamic IGF-I could be a response to the increased GH levels (13, 42). The present results also demonstrate that the increased expression of GHRF-R and GHS-R by the absence of liver-derived IGF-I is not reversed by the enhanced serum GH levels or the enhanced hypothalamic IGF-I expression.

The decrease in circulating IGF-I and increased expression of pituitary GHRF- and GHS-Rs was accompanied by enhanced GHRF- and GHS-induced GH secretion in vivo. Therefore, endogenous, liver-derived IGF-I exerts a GHRF antagonistic effect similar to that originally shown in rat pituitary cells in vitro (9). IGF-I infusion to humans leads to decreased GH response to GHRF treatment in fed men, but not women, in one study (5), but in another study, IGF-I treatment did suppress both GHRF- and GHS-induced GH secretion in fasted young women (43). In the present study, the effect of liver IGF-I depletion on GHS responsiveness was more pronounced in female than in male mice, although the GHS-R expression was enhanced to a similar degree in both sexes. These results suggest that mechanisms other than receptor expression may affect GHS responsiveness. Taken together, the results of the present and previous data indicate that liver-derived IGF-I exerts a feedback-regulation of GH secretion by suppression of GHRF-R and GHS-R expression at the pituitary level. These enhanced receptor levels and other, as yet unknown mechanisms may then decrease sensitivity to ligand stimulation.

It was suggested already in the 1980s that the masculinizing effects of the male GH secretion pattern could be dependent on hepatic IGF-I production (18). It was shown that continuous exposure to GH can feminize the expression of MUP and PRL-R in the livers of male mice (18). In the present study, liver-specific IGF-I depletion indeed caused a demasculinization of liver functions, and the overall distribution of GH levels in LI-IGF-I-/- mice was changed from lower to higher values. A simple interpretation of these data combined is that the feminization of hepatic functions is caused by an increase in the low basal GH levels normally found in male rodents (2, 44). The present data do not provide information on whether GH pulse height was enhanced. Because serum IGF-I levels were decreased by 80% in the LI-IGF-I-/- mice, it thus appears that the well-documented sexual dimorphism of hepatic functions induced by the GH-secretion pattern (1, 20) can be influenced by a feedback signal from the liver. Both male and female LI-IGF-I-/- mice in this study had increased relative liver weight, in line with earlier pooled male and female data (22). It is reasonable to hypothesize that the increased liver weight in the LI-IGF-I-/- mice also is due to the increased GH levels because GH can also affect relative liver size (45, 46). Taken together, these results are consistent with a pituitary-liver feedback axis that is more important for regulation of liver functions than it is for body growth.

In conclusion, loss of liver-derived IGF-I feedback on the hypothalamic-pituitary system increases GH secretion in both male and female mice (see proposed model in Fig. 6Go), which, in turn, stimulates liver growth. Moreover, elevated GH troughs in male mice with IGF-I knockout leads to feminization of GH-regulated sexually dimorphic liver functions. Our data show that depletion of liver-derived IGF-I increases the expression and sensitivity of pituitary GHRF and GHS receptors. Therefore, we conclude that the major site of action of liver-derived IGF-I in the regulation of GH secretion is at the pituitary rather than at the hypothalamic level.



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  		Figure 6. A proposed model of a liver-pituitary feedback axis and how it is affected by depletion of liver-derived circulating IGF-I. Loss of liver-derived IGF-I feedback on the hypothalamic-pituitary system increases GH levels, including trough levels (and possibly also pulse height). This in turn increases liver weight in both genders and feminizes liver functions (MUP and PRL-R expression) in male liver-specific IGF-I knockout mice. Our data indicate that increased expression of pituitary receptors for GHRF and GHS increases the sensitivity to GHRF and GHS in the IGF-I-depleted mice. Therefore, the major site of action of liver-derived IGF-I in the feedback regulation of GH-secretion is at the pituitary rather than at the hypothalamic level.

 


   Acknowledgments
 
We thank Danielle Carmignac and Professor Iain Robinson for valuable help with GH measurements. We are grateful to Dr. Derek LeRoith for providing the mice with loxP sequences flanking exon 4 of the IGF-I gene, Dr. Ralph Kühn and Professor Claus Rajewsky for providing the Mx-Cre mice, Professor Charles Weissmann for interferon-{alpha}2/{alpha}1 and Dr. Ian Ahnfelt-Rönne and Dr. John Römer at Novo Nordisk A/S for providing Ipamorelin. We thank Maud Pettersson, Department of Clinical Pharmacology, for valuable technical assistance. The intracellular region PRL receptor probe was a kind gift from Kåre Hultén.


   Footnotes
 
This work was supported by the Swedish Medical Research Council (0998), the European Union (Framework 5, QLRT-1999-02038), the Swedish Foundation for Strategic research, the Bergvall foundation, the Lundberg Foundation, the Nordic Insulin Pharma, the Swedish Medical Society, the Göteborg Medical Society, Pharmacia-Upjohn, Novo Nordisk Foundation, the Swedish Association Against Rheumatic Disease, the Adlerbertska Research Foundation, the Sahlgrenska University Foundation, the Foundation of Ragnar and Torsten Söderberg, USPHS Grant DK-30667 (to R.D.K.), and the Bane Foundation (to L.A.F.).

Abbreviations: GAPDH, Gyceraldehyde-3-phosphate dehydrogenase; GHRF, GH releasing factor; GHRF-R, GHRF receptor; GHS, GH secretagogues; GHS-R, GH secretagogue receptor; LI-IGF-I-/-, liver-specific IGF-I knockout; MUP, major urinary protein; PRL-R, PRL receptor; RPA, ribonuclease protection assay; sst, somatostatin receptor.

Received June 6, 2001.

Accepted for publication July 19, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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