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Endocrinology Vol. 138, No. 3 1066-1071
Copyright © 1997 by The Endocrine Society

Articles

Basal and Growth Hormone-Induced Hepatic Messenger Ribonucleic Acid Expression of Insulin-Like Growth Factor-I (IGF-I) and IGF-Binding Protein-3 Is Independent of Hyperinsulinemia and Increased Energy Status in the Genetically Obese Zucker Rat1

E. Melián, B. Velasco, R. Barrios and F. Sanchez-Franco

Servicio de Endocrinología, Centro Nacional de Investigacion Clinica, Instituto de Salud Carlos III (E.M., B.V., F.S-F.); and Servicio de Pediatría, Hospital Ramón y Cajal (R.B.), Madrid, Spain

Address all correspondence and requests for reprints to: Elvira M. Melián Pérez, Servicio de Endocrinología, Centro Nacional de Investigacion Clinica, Instituto Carlos III, C/Sinesio Delgado, 10, 28029 Madrid, Spain.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Genetically obese Zucker rats, like obese humans, have normal or elevated circulating insulin-like growth factor-I (IGF-I) levels in the presence of low GH secretion. Hyperinsulinemia, increased energy status, or other nutritional factors associated with obesity could be responsible for these findings directly by increasing hepatic IGF-I production at the transcriptional or posttranscriptional level. Alternatively, circulating IGF-I could be modulated indirectly by affecting its binding proteins. To further elucidate this point, we quantitated hepatic IGF-I, IGF binding protein-3 (IGFBP-3), and GH receptor messenger RNAs (mRNAs) expression in obese Zucker rats under different serum GH and insulin conditions using lean rats as controls. Eleven-week-old male rats were studied basally (intact) or after hypophysectomy (hx) at 9 weeks. In each condition, animals were killed before or 6 h after one dose of recombinant human GH (1.5 µg/g body weight ip). At this time, in addition to the mRNA expression of the above-mentioned genes, body weight, glycemia, insulinemia, serum GH (rat and human), and serum IGF-I levels were determined. Obese Zucker rats were significantly heavier than controls in all the conditions studied and did not show differences in glycemia. Severely hyperinsulinemic intact obese rats (146.9 ± 14 vs. 46.3 ± 3 µU/ml, P < 0.001) showed compared with intact lean rats significantly lower serum GH (2.39 ± 0.9 vs. 4.98 ± 0.68 ng/ml, P < 0.01), decreased hepatic IGF-I mRNA and IGFBP-3 mRNA accumulation (IGF-Ia: 79 ± 5.9% vs. 100 ± 0.9%, P < 0.05; IGF-Ib: 67 ± 5.5% vs. 100.1 ± 1.9%,P < 0.001; IGFBP-3: 54.7 ± 2.75% vs. 100.5 ± 1.55%, P < 0.001), and similar circulating IGF-I levels (1439 ± 182 vs. 1516 ± 121 ng/ml). Under comparable serum GH levels in GH-treated intact, hx, and GH-treated hx animals, hyperinsulinemia and/or increased body weight present in obese rats were not associated with increased hepatic IGF-I and IGFBP-3 mRNA amount. No differences in GH receptor/GH-binding protein mRNAs were found in any experimental condition. These results suggest that in vivo the imbalance of the serum GH/IGF-I axis present in obesity is primarily due to events distal to the hepatic IGF-I and IGFBP-3 mRNAs expression, which is tightly correlated to GH levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor-I (IGF-I) is an important regulator of animal growth and is thought to mediate many of the physiological actions of GH (1). The liver has the greatest abundance of IGF-I messenger RNA (mRNA), and hepatic synthesis of IGF-I can account for the known turnover of this peptide in the circulation (2). Total hepatic IGF-I expression is regulated both by GH and nutritional status in a complex and unelucidated way that links diet and growth (1, 3, 4). Thus, although diminished GH secretion is a well-established feature of obesity, children with idiopatic obesity have normal or rapid rates of linear growth and normal or increased IGF-I plasma levels (5). Normal or elevated serum levels of IGF-I have been reported also in obese adults (6) and in the obese Zucker rat, an animal model of obesity with hyperinsulinemia and low GH (7). Chronically increased energy status, hyperinsulinemia, and other nutritional factors associated with obesity could be responsible for the serum GH/IGF-I axis imbalance, acting directly on the hepatic IGF-I gene at a transcriptional or posttranscriptional level, or modulating its binding proteins (3, 4). Of those factors, a relevant role of hyperinsulinemia in the stimulation of hepatic IGF-I production has been suggested, based on the fact that insulin increases hepatic IGF-I mRNA transcription in primary cultures of hepatocytes, even in the absence of GH (8, 9).

The rat and human IGF-I genes contain six exons. The mature protein is encoded by exons 3 and 4, and the last one also contains the N-terminal portion of the so-called E peptide, which appears in the unprocessed hormone. Two molecular mechanisms of control of IGF-I gene expression have been described in rats and humans. The first is the existence of several start sites for transcription in leader exons 1 and 2 encoding for different 5'-untranslated regions and amino-terminal extensions of putative IGF-I signal peptides (10). The second is the alternative splicing of the primary IGF-I transcript involving exons 5 and 6, resulting in two different E peptides depending on whether exon 4 is spliced to exon 5 (Eb region; IGF-Ib) or exon 6 (Ea region; IGF-Ia) (11). E peptides have been implicated in IGF-I mRNA stability (12), and some authors suggested a specific role for the Eb mRNA in directing IGF-I to the circulation on the basis of its postnatal presence, mainly in the liver, and its higher responsiveness to the administration of GH in hypophysectomized rats (13).

The aim of the present study was to investigate in an obesity model in vivo the effects of hyperinsulinemia and chronically increased energy status on hepatic IGF-I production under different GH conditions. Using age-matched lean rats as controls, we examined both hepatic IGF-I mRNA trancripts (IGF-Ia and IGF-Ib) amount and ratio, IGF-binding protein-3 (IGFBP-3) mRNA, GH receptor (GHr) and GH binding protein (GHBP) mRNAs, and serum IGF-I in intact and hypophysectomized (hx) obese Zucker rats before or 6 h after acute GH treatment. Whereas intact obese rats remained severely hyperinsulinemic (14), hypophysectomy enabled us to decrease insulin levels in rats that still had an increased body weight (15). We added fasting for 18–24 h to the hx groups to further lower insulin levels and to avoid acute nutritional-induced modulations of insulin and IGF-I levels (4).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
Intact rat protocol. Male lean (Fa/-) and genetically obese (fa/fa) rats were obtained from Criffa (Barcelona, Spain). Animals were fed a standard chow (RMN Labsure, Biosure, Barcelona, Spain) and water ad libitum and were housed at constant temperature (23 C) with a fixed (12 h light. 12 h dark) light cycle. After adaptation, intact animals were anesthesized and killed by decapitation at 0900 h (lean, n = 6; obese, n = 6) or injected with one ip dose of 1.5 µg/g body weight of recombinant human GH (rhGH) (Norditropin, Novo-Nordisk, Bagsvaerd, Denmark) and killed 6 h later (lean, n = 5; obese, n = 5). Blood was collected from the cervical vessels, and serum was stored at -20 C until assayed for glycemia, insulinemia, GH (rat or human), and IGF-I. Livers were removed rapidly, snap-frozen in dry ice, and stored at -80 C. At the time of the experiments, animals were 11 weeks old.

Hypophysectomized rat protocol. Male lean and genetically obese rats were delivered to our animal quarters 7 days after hypophysectomy (Criffa, Lyon, France). The adequacy of hypophysectomy was assessed at the time of killing by visual inspection of sella turcica. Rats were housed for 5 days under controlled conditions with ad libitum feeding and fluid (water with 5% glucose and 0.9% NaCl). After adaptation, rats were fasted for 18 h and decapitated at 0900 h (lean, n = 5; obese, n = 5) or injected with one ip dose of 1.5 µg/g body weight of rhGH and decapitated 6 h later, i.e. after 24 h of fasting (lean, n = 5; obese, n = 5). At the time of the experiments, animals were 11 weeks old. Serum and liver were collected and stored at -20 C and -80 C, respectively.

RIAs and biochemical parameters
IGF-I was measured by a commercial RIA (Nichols Institute, San Juan Capistrano, CA) after acid ethanol extraction. Rat GH was determined using the National Pituitary Hormone Distribution Program rat hormone kit (NIAMDD, Bethesda, MD) with a sensitivity limit of 0.8 µg/liter. Serum insulin was measured using a commercial kit (Coat-a-Count Insulin, Diagnostic Products Corp., Los Angeles, CA). Glucose was assayed in plasma by the glucose oxidase method using a commercial kit (ITC Diagnostics).

All the parameters were measured in serum from individual rats, and all samples where a comparison was made were analyzed in the same assay to avoid interassay variations.

RNA probes
IGF-I. One construct of 376 bp from the rat IGF-I complementary DNA (cDNA), containing part of the A domain, the entire D and E domains, and part of the 3'-untranslated region was generated to simultaneously quantify mRNAs with (IGF-Ib: 376 bp) and without (IGF-Ia: 224 bp) the 52-bp insert present in the E domain of some IGF-I mRNAs. For the protection assay, this template was linearized with HindIII and transcribed with T7 RNA polymerase (16).

GHr. The rat GHr probe was transcribed from a 900-bp BgII fragment of a rat GHr cDNA corresponding to the region encoding the signal peptide, the extracellular domain, the transmembrane domain, and a portion of the intracellular domain (17). This template was linearized with BamHI and transcribed with T7 RNA polymerase to generate a 445-base antisense RNA probe. This probe yields two protected bands when hybridized to total liver RNA, a 439-base band corresponding to the GHr mRNA, and a 290-base band corresponding to the alternately spliced mRNA which, in the rat, encodes the GHBP.

Cyclophilin. The rat cyclophilin cDNA was a 132-bp fragment linearized with APAI and transcribed to generate the antisense probe with SP6 polymerase following previously described methods (18).

IGFBP-3. The rat IGFBP-3 probe corresponds to nucleotides of the rat cDNA clone described by Albiston et al. (19).

Ribonuclease protection assay
Liver RNA was extracted using the Chomczynski and Sacchi method (20). In the ribonuclease protection assay total RNA of each individual rat was hybridized overnight with approximately 600,000 cpm of labeled antisense rat IGF-I or GHr riboprobe at 45 C. The hybridization solution contained 75% (vol/vol) formamide, 80 mM Tris HCl, pH 7.6, 4 mM EDTA, 1.6 M NaCl, and 0,4% SDS. After hybridization, samples were digested using RNase A (40 µg/ml) and RNase T1 (2 µg/ml) for 1 h at 30 C. Protected hybrids were isolated by ethanol precipitation after phenol-chloroform extraction and separated according to size on an 8% polyacrylamide/8 M urea denaturing gel. Gels were exposed to x-ray film (Kodak, Cambridge, U.K.) at -80 C for 24–36 h. Quantitation of the intensities of the autoradiography bands corresponding to protected hybrids was done by densitometric scanning using Adobe-Photoshop 2.0 and NIH-Image 1.47 programs for the Apple Macintosh (Cupertino, CA). All samples were hybridized at the same time with cyclophilin to correct for the differences in gel loading.

Northern analysis
Total RNA was electrophoresed in a 1% agarose-0.66 M formaldehyde gel, followed by electrotransfer to nylon membrane (Nytran, Schleicher & Schuell, Keene, NH) and UV cross-linking (Hoefer Scientific Instrument, San Francisco, CA). Membranes were prehybridized and hybridized for 24 h at 65 C in 50% formamide with approximately 600,000 cpm/ml of [32P]uridine triphosphate (800 Ci/mmol) labeled antisense rat IGFBP-3 probe. Autoradiograms and quantitation of intensities were done as described above. Equal loading was confirmed by comparing intensities of ethidium bromide-stained ribosomal 28S RNA in the nylon filter.

Statistical analysis
The statistical significance of differences between values was calculated by unpaired Student’s t test and/or variance analysis. The differences were considered statistically significant when P-values were below 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum parameters and body weight
Body weight, glycemia, and insulinemia in each experimental group at the time of death are summarized in Table 1Go. Obese rats represented an appropiate model of increased energy storage, remaining significantly heavier than controls in the four experimental conditions of the study. Significant hyperinsulinemia was present in intact and GH-treated intact obese rats and disappeared in the hx and 18 h-fasted obese animals. After rhGH treatment, however, obese rats had significantly higher insulin levels than lean controls. Serum glucose levels were not significantly different between phenotypes at any condition.


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  		Table 1. Body weight and serum parameters in intact, GH-treated intact, hx, and GH-treated hx lean and obese Zucker rats

 
Serum GH (rat or human) and IGF-I levels in the four experimental manipulations are also shown in Table 1Go. Intact obese Zucker rats showed similar serum IGF-I when compared with lean animals in the presence of significantly reduced levels of circulating rGH. We found no differences in serum GH (rat or human) or IGF-I between obese and lean animals in any other experimental condition. Overall, hx and fasted rats of both groups had serum IGF-I under the detection limit of the assay and higher and measurable levels in the rhGH-treated rats, in spite of the 24-h fast.

Liver IGF-I mRNA in intact and GH-treated intact lean and obese Zucker rats
We first studied whether the manifest hyperinsulinemia present in intact obese rats was associated with an increased amount of hepatic IGF-I mRNA in relation to GH levels, which could explain serum IGF-I findings. Figure 1Go shows IGF-I mRNA levels in intact and GH-treated intact lean and obese rats. These were decreased in intact obese rats compared with intact lean rats, and this decrement affected both transcripts, although it was more evident for IGF-Ib (IGFIa: 79 ± 5.9% vs. 100 ± 0.9%, P < 0.05; IGFIb: 67.13 ± 5.5% vs. 100.1 ± 1.9%, P < 0.01). Hyperinsulinemic GH-treated obese rats showed similar IGF-I expression to GH-treated lean controls (compared with intact lean: IGF-Ia, 111 ± 3.4% lean and 112.3 ± 1.5% obese; IGF-Ib, 142.1 ± 16% lean and 130 ± 16% obese). According to these data IGF-b/total IGF-I ratio was significantly decreased in intact obese animals and similar to the lean group in the GH-treated intact obese rats (Table 2Go).



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  		Figure 1. IGF-I mRNA in livers of intact and GH-treated intact lean and obese Zucker rats. Twenty micrograms of total liver RNA from lean or obese Zucker rats were subjected to solution hybridization/RNase protection assay using antisense probes as described in Materials and Methods. Positions of each protected fragment are indicated on right. To visualize both IGF-I transcripts, the gel was exposed for 18 h in the case of IGF-Ia and for 72 h in the case of IGF-Ib. After correction for cyclophilin levels, optical density units were adjusted so that the ratio obtained from livers of intact lean rats equaled 100. Results are mean ± SEM (n = 4–5).*, P < 0.05; **, P < 0.01 vs. lean rats in same experimental condition. O.D.U., optical density units.

 

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  		Table 2. IGF-Ib/IGF-Ia + b and GHr/GHr + GHBP in intact, GH-treated intact, hx, and GH-treated hx lean and obese Zucker rats

 
Liver IGF-I mRNA in hx and GH-treated hx lean and obese Zucker rats
We were also interested in establishing whether the excess weight of obese rats could positively modulate the amount of hepatic IGF-I mRNA compared with lean rats when serum GH and insulin levels were comparably suppressed. As shown in Fig. 2Go, we found no differences between hx rats before (IGF-Ia: 126 ± 13% lean vs. 100 ± 10% obese; IGF-Ib: indetectable) or after rhGH, with the exception of a slightly but significant decrease in the IGF-Ib transcript after rhGH in the obese rats (IGF-Ia vs. hx lean: 690 ± 60% lean and 700 ± 54% obese; IGF-Ib: 100 ± 1.4% lean vs. 89 ± 5.7% obese, P < 0.05). Overall liver IGF-I mRNA levels were markedly reduced in hx fasted animals (compare Hx vs. I), the IGF-Ia transcript showing a 6- to 8-fold decrease, and the IGF-Ib transcript being almost indetectable. However both messages were massively expressed in the GH-treated groups in spite of low insulinemia and 24 h of fasting (compare Hx + GH vs. I).



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  		Figure 2. IGF-I mRNA in livers of intact (I), hx, and GH-treated hx lean and obese Zucker rats under the conditions described in legend of Fig. 1Go. To visualize both IGF-I transcripts, the gel was exposed for 18 h in the case of IGF-Ia and for 26 h in the case of IGF-Ib. After correction for cyclophilin levels, ratios obtained from livers of hx lean rats for IGF-Ia and from livers of GH-treated hx lean rats for IGF-Ib were used as 100. Results are mean ± SEM (n = 5). *, P < 0.05 vs. lean rats in same experimental condition.

 
Liver GHr and GHBP mRNA in intact, hx, and GH-treated hx lean and obese Zucker rats
No significant changes were observed in GHr and GHBP mRNA levels, and their ratios between the two rat phenotypes in any experimental condition (Fig. 3Go; Table 2Go).



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  		Figure 3. GHr and GHBP mRNAs in livers of intact, hx, and GH-treated hx lean and obese Zucker rats under the conditions described in legend of Fig. 1Go. After correction for cyclophilin levels, O.D.U. were adjusted so that the ratio obtained from livers of intact lean rats equaled 100. Results are mean ± SEM (n = 5).

 
Liver IGFBP-3 mRNA in intact, hx, and GH-treated hx lean and obese Zucker rats
Because the presence of IGFBP-3 in serum plays an important role in prolonging the half-life of IGF-I, we evaluated a possible effect of insulinemia and/or energetic factors on the amount of hepatic IGFBP-3 mRNA accumulated in obese rats compared with lean controls. As shown in Fig. 4Go, intact obese rats showed a decreased amount of IGFBP-3 mRNA when compared with intact lean animals (54.7 ± 2.75 vs. 100.5 ± 1.55, P < 0.01). No significant differences were observed between hx groups before or after rhGH treatment. Overall, hepatic IGFBP-3 amount was intensely reduced in hx fasted animals, and in-between levels of expression were present in the hrGH-treated hx rats.



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  		Figure 4. IGFBP-3 mRNA in livers of intact, hx, and GH-treated hx lean and obese Zucker rats. Twenty micrograms of total liver RNA from lean or obese Zucker rats were subjected to Northern blot using IGFBP-3 probe as described in Materials and Methods. After correction for 28S levels, O.D.U. were adjusted so that the ratio obtained from livers of intact lean rats equaled 100. Results are mean ± SEM (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Obesity in the Zucker rat is inherited as a recessive gene mutation, as yet unidentified, localized in chromosome 5 (21). Compared with lean littermates, the homozygous (fa/fa) rats show extreme obesity from a young age, insulin resistance with hyperinsulinemia, normal glycemia or mild hyperglycemia, and hyperlypidemia (14). Moreover, these animals develop GH deficiency as early as 6 weeks of age (22). It has been shown that plasma levels of GH, pulsatile GH release, pituitary GH concentration, and mRNA expression are reduced in adult obese male rats when compared with lean littermates, and that this defect is maximal at 10–12 weeks of age (23, 24, 25, 26). These conditions make this rat a good experimental model of early-onset human obesity.

Although early studies of plasma somatomedin by bioassay found low values of IGF-I in obese animals (27), more recent reports have shown normal to high levels of this peptide in plasma measured by RIA (26, 28, 29). Recently, Nguyen-Yamamoto et al. (7) showed that obese Zucker rats have normal or increased rates of linear growth accompanied by normal or increased circulating IGF-I in a broad spectrum of ages in both sexes. These authors even found an increased plasma response to exogenous GH in females compared with lean controls (7). We found similar serum IGF-I levels in intact rats of both phenotypes in spite of GH differences. In GH-treated intact and hx animals, the obese group showed relatively higher serum IGF-I levels after rhGH administration, but dispersion of the data precluded the establishment of significant differences.

In our study absolute serum IGF-I values were slightly higher than those found by Nguyen-Yamamoto et al. (7) in intact male Zucker rats of the same age. These differences could be due to the affinity of the antibody used for the RIA, or to the efficiency of the extraction procedure to remove IGFBPs (30, 31). In support of the last possibility, we have recently observed with two different anti-IGF-I antisera, that the addition of cryoprecipitation after acid-ethanol extraction, which was used by the afore-mentioned investigators, greatly reduces the interference from binding proteins and decreases final IGF-I values in rat serum.

It has been suggested that GH-independent growth of the obese Zucker rats could result from direct effects of hyperinsulinism on circulating levels of IGF-I and/or IGFBP-3 production and/or indirect effects through increased GHr function (7). Our study has established the relationship between hepatic IGF-I mRNA expression and serum IGF-I peptide modifications under different GH and insulinemic conditions in this obesity model. Our results suggest that hepatic IGF-I mRNA accumulation in vivo is tightly correlated to GH levels in obese rats and that modulation of serum IGF-I levels in this obesity model may occur at a posttranscriptional level. Thus, compared with lean controls, severely hyperinsulinemic and GH-deficient intact obese rats showed decreased liver IGF-I mRNA with no alteration of circulating IGF-I. Additionally, hyperinsulinemia and/or increased body weight of obese rats were not associated with higher hepatic IGF-I mRNA levels in any of the experimental conditions with comparable serum GH levels: GH-treated intacts, hx, and GH-treated hx rats. This last group even showed a decreased amount of IGF-Ib transcript of unexplored implications. The presence of similar levels of GHr mRNA amount between lean and obese rats would not exclude differences in the receptor functionality induced by insulin. Nevertheless, hepatic IGF-I mRNA findings do not support a physiological relevance for them, because they were not accompanied by increased IGF-I amount in the obese animals at any condition.

Insulin could increase circulating IGF-I levels in GH-deficient rats by increasing IGFBP-3 production which, in turn, would maintain serum IGF-I levels. In fact, insulin is able to stimulate IGFBP-3 in cell cultures (32), and this binding protein is present in normal amounts in the serum of obese Zucker rats (7). In our in vivo study, we found a significant decrease in IGFBP-3 mRNA in low GH and hyperinsulinemic intact obese rats compared with controls, suggesting that its modulation would probably occur, the same as for IGF-I, at a posttranscriptional level. In agreement with this hypothesis, we found no differences in IGFBP-3 mRNA amounts between lean and obese rats with comparable serum GH conditions. Previous studies in normal rats have shown that changes in circulating IGFBP-3 appear not to be directly related to changes in its hepatic mRNA levels, suggesting that posttranscriptional events must play an important role over serum IGFBP-3 (33).

Obesity-related hyperinsulinemia could increase free IGF-I by reducing the concentration of circulating IGFBP-I, because insulin effects as a suppressor of hepatic IGFBP-I production are not affected by metabolic insulin resistance (34). We did not measure hepatic IGFBP-I expression in our experimental conditions, but serum IGFBP-I levels are supressed by 67% vs. controls in 24 h-fasted male obese Zucker rats (35). Accordingly, it has been shown recently (36) that in human obesity there is an increase in free IGF-I, without an alteration in total IGF-I levels, showing direct correlation with hyperinsulinemia and inverse correlation with IGFBP-I levels (36).

The IGF-Ib transcript levels appear to be more tightly regulated by GH compared with IGF-Ia mRNA levels. This is reflected in the more significant decrement of IGF-Ib/total IGF-I ratio observed in intact GH-deficient obese rats (Table 2Go). Indeed, several experiments support that the rat hepatic IGF-Ib mRNA variant of the IGF-I gene may be more sensitive to GH and energy status (16, 37). It has been suggested that the differential regulation of exon 5 could be linked to separate 5'-leader exons and be responsible for programmed endocrine or paracrine fate of hepatic IGF-I, but there is little evidence to support this at present (13). E peptide could have specific effects by itself, at least in humans, where it is present in serum. Part of the Eb peptide contains amidated growth-promoting portions with specific binding sites in human tissues (38, 39).

Finally, the ability of hx and 24 h-fasted rats to elicit a remarkable response of liver IGF-I mRNA to high doses of exogenous rhGH suggest that the machinery involved in the IGF-I gene transcription is intact in spite of fasting and other hormonal deficiencies, and supports the importance of translational and posttranslational effects in the IGF-I regulation by the nutritional status (40).

In summary, this study shows that genetically obese and hyperinsulinemic Zucker rats, compared with lean littermates, have 1) low hepatic IGF-I and IGFBP-3 mRNA expression with normal circulating levels of IGF-I in the presence of low GH; 2) comparable hepatic IGF-I and IGFBP-3 mRNA expression in conditions of serum GH equivalence; and 3) similar GHr/GHBP mRNA levels in all conditions studied. We conclude that in vivo the regulation of endocrine IGF-I production by energetic/nutritionals factors associated with obesity probably occurs at a distal point to the hepatic IGF-I and IGFBP-3 mRNAs expression, which seems to be basically GH dependent.


   Acknowledgments
 
The authors wish to thank Dr. S. Lamas for helpful comments and Dra. L. Cacicedo for her support. Drs. E. Hernandez, D. LeRoith, and S. Ojeda are gratefully acknowledged for providing the cDNAs necessary to generate the riboprobes. We also thank Purification Mota for her technical assistance.


   Footnotes
 
1 This work was supported by a grant from the Spanish FIS (Fondo de Investigaciones Sanitarias) 94/0355. Back

Received November 6, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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