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(The FASEB Journal. 1998;12:1455-1460.)
© 1998 FASEB

RESEARCH COMMUNICATION

Transgenic rabbits overexpressing growth hormone develop acromegaly and diabetes mellitus

Cristina Costaa, Gemma Solanesa, Joana Visaa, and Fatima Boscha,1

a Departament de Bioquimica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Transgenic rabbits expressing the bovine growth hormone (bGH) gene in liver and kidney were obtained to study the long-term effects of chronic exposure to GH in nonrodent animals. These rabbits presented high levels of bGH and insulin-like growth factor I in serum. In spite of chronic exposure to bGH, transgenic rabbits had similar body weight to controls. However, enlargement of the head and limbs and reduction of visceral fat were observed in these animals. They also showed marked hyperinsulinemia, hyperglycemia, and hypertriglyceridemia, indicating that they developed insulin resistance. Furthermore, serious histopathological alterations, including marked fibrosis, were observed in liver, kidney, and skeletal muscle. These anatomical, metabolic, and histological alterations closely resemble those found in patients with acromegaly. Thus, transgenic rabbits overexpressing GH may be a good model of the human disease.—Costa, C., Solanes, G., Visa, J., Bosch, F. Transgenic rabbits overexpressing growth hormone develop acromegaly and diabetes mellitus. FASEB J. 12, 1455–1460 (1998)


Key Words: bovine growth hormone • IGF • PEPCK • hyperglycemia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
GROWTH HORMONE (GH)2 exerts a broad variety of biological actions promoting body growth and regulating carbohydrate, lipid, and protein metabolism (1). Its effects may be direct or indirect by inducing insulin-like growth factor I (IGF-I) release from the liver into the bloodstream or the local production of IGF-I in different organs (2, 3). Furthermore, GH has been described to act directly on pancreatic ß-cells to stimulate insulin secretion and ß-cell proliferation (4). In addition, GH has anti-insulin activity, which corresponds to chronic effects of the hormone (1, 5). Chronic effects of GH are characterized by a pattern of insulin resistance in a number of parameters, including hepatic and muscle glycolysis and glycogenesis, hepatic gluconeogenesis, and lipid metabolism (6).

Acromegaly is a chronic disease in which prolonged elevation of GH levels after puberty leads to excessive tissue growth, multiple disturbances in other hormonal systems, and altered intermediary metabolism (5). Major physical and histological alterations found in acromegalic patients include bony overgrowth of the skull, enlargement of the extremities, and connective tissue abnormalities (such as overgrowth and fibrosis) (7). Treatment strategies include surgery, radiation therapy, and pharmacotherapy; however, only about 60% of patients are curable by a surgical approach, and other therapies have lower success rates (8).

Acromegaly is usually confirmed by an increase in GH levels, lack of GH suppression during an oral glucose tolerance test, and an elevated level of IGF-I (9), which has been shown to correlate better with clinically active acromegaly than the basal or postglucose GH levels (10). Glucose intolerance and insulin resistance are highly prevalent (about 60%) in patients with acromegaly (11, 12). Insulin resistance is manifested by striking hyperinsulinemia in response to oral or intravenous glucose and other secretagogues (12). Overt diabetes is frequent and severe in patients with high GH levels (13).

Although researchers have developed and extensively studied several transgenic mouse lines overexpressing GH (14–16), these animals are not suitable models of human acromegaly, because they show a giant phenotype that is not observed in the human disease. In addition, transgenic pigs overexpressing GH do not seem to show acromegalic sclerotic alterations, although they develop severe alterations such as arthritis and stomach ulcers (17–19). With this study, we sought to develop a nonrodent animal model to determine long-term effects of exposure to GH. To this end, we generated transgenic rabbits expressing a P-enolpyruvate carboxykinase/bovine growth hormone (PEPCK/bGH) chimeric gene (16) and found that these rabbits showed metabolic, histological, and anatomical alterations that closely resembled those found in patients with acromegaly. This study also suggests that transgenic rabbits overexpressing GH might be a useful model to assay new therapies for acromegaly.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Generation of transgenic rabbits
Transgenic rabbits were generated by microinjection of a PEPCK/bGH chimeric gene (16), kindly provided by Dr. R. W. Hanson (Case Western Research University), into rabbit zygotes. This chimeric gene contains the bGH gene under control of the -450 bp to +73 bp fragment of the PEPCK promoter, which has been shown to direct the expression of foreign genes to the liver and kidney of transgenic mice (16). Fertilized oocytes were collected by flushing the oviducts of donor New Zealand hybrid rabbits 19 h after mating (20). About 2–3 pl of DNA solution (10 ng/µl) was injected into the zygote pronucleus. The injected zygotes were transferred through the fimbrial end of the oviduct (about 10 zygotes per side) of a pseudopregnant foster mother. At 4 days of age, the animals were tested for the presence of the transgene by Southern blot of 10 µg of ear DNA digested with PvuII. Blots were hybridized with the entire PEPCK/bGH chimeric gene radiolabeled with [{alpha}-32P]dCTP ((3000 Ci/mmol), Amersham Corp., Arlington Heights, Ill.) by random oligopriming (Boehringer-Mannheim, Mannheim, Germany).

Transgenic and control rabbits were weighed at weaning (1 month of age), fed a growth diet (16.5% protein, Corena, Reus, Spain), and weighed at 2 months of age.

RNA analysis
Total RNA was prepared from animal tissues by the guanidine isothiocyanate method (21) and RNA samples (30 µg) were electrophoresed in a 1% agarose gel containing 2.2 M formaldehyde. Northern Blot was hybridized to 32P-labeled bGH probe (1.3 kb fragment obtained by digestion of the bGH gene with PvuII). This probe was labeled using [{alpha}-32P]dCTP, following the method of random oligopriming described by the manufacturer. Specific activity of the DNA probe was approximately 109 cpm/µg DNA. Membranes were placed in contact with Kodak XAR-5 films.

Hormone and metabolite determinations
Blood samples were obtained from control and transgenic rabbits from the ear vein between 9:00 and 10:00 AM. Serum bGH was measured by enzyme-linked immunosorbent assay using a specific anti-bovine GH antibody (obtained from the National Hormone and Pituitary Program) (16). IGF-I was measured by radioimmunoassay (RIA) after acid-ethanol extraction from plasma using an anti-human IGF-I antibody (Nichols Institute Diagnostics, San Juan Capistrano, Calif.). Insulin was also measured by RIA (CIS, Biointernational, Gif-Sur-Yvette, France). Serum glucose (Glucoquant, Boehringer-Mannheim), triglycerides (GPO-PAP, Boehringer-Mannheim), and cholesterol (Monotest, Boehringer-Mannheim) were determined enzymatically.

Histopathological analysis
Tissue samples were fixed for 24 h in 10% buffered formalin, embedded in paraffin, and sectioned (2–3 µm). Sections of skeletal muscle were stained with hematoxylin/eosin. Kidney sections were stained with periodic acid-Schiff and counterstained with hematoxylin. Liver sections were stained with Masson trichromic reagent.

Statistical analysis
Values obtained from each transgenic rabbit were compared with values obtained from a population of control rabbits of the same age. All values obtained in control rabbits are expressed as the means ±SEM. Statistical analysis was carried out using analysis of variance. Differences were considered statistically significant at P < 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
To study long-term effects of exposure to GH, a PEPCK/bGH chimeric gene (16) ( Fig. 1A) was microinjected into the pronucleus of rabbit zygotes to obtain transgenic rabbits. Two transgenic female rabbits were obtained that had incorporated the chimeric gene as confirmed by Southern blot analysis (Fig.1B). After hybridization of the membranes, three diagnostic bands of 1.3, 0.6, and 0.3 kb were detected in transgenic rabbits and were not present in controls ( Fig. 1B). Transgenic rabbit 1 (Tg1) was estimated to carry about two copies of the transgene, whereas transgenic rabbit 2 (Tg2) had incorporated about five copies of the transgene when compared with known amounts of the chimeric gene (data not shown). These two founder females did not reproduce after several matings, probably because the expression of the transgene affected reproductive function. These animals showed anestrus and a general hypoplasia of the reproductive tract (data not shown). Histological examination of the ovaries of these transgenic animals revealed numerous atretic follicles compared to ovaries from control rabbits and no follicles of Graaf (data not shown). Similarly, transgenic pigs expressing bGH do not show estrus, and their ovaries are devoid of corpora lutea and corpora albicantia (18). Reproductive abnormalities have also been reported in acromegaly (e.g., menstrual disorders and decreased libido) (13).



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  		Figure 1. Generation of transgenic rabbits expressing the PEPCK/bGH chimeric gene. A) Schematic representation of the PEPCK/bGH chimeric gene (16). The size of the fragments obtained by PvuII digestion is indicated. B) Southern Blot analysis of genomic DNA obtained from control and Tg1 and Tg2 transgenic rabbits was performed as indicated in Materials and Methods. Blots were hybridized with the entire PEPCK/bGH chimeric gene. Lanes 1–4, 6, 7, control rabbits; lane 5, Tg1; lane 8, Tg2. C) Northern Blot analysis of total RNA obtained from liver and kidney of control and transgenic rabbits was performed as indicated in Materials and Methods. A 1.3 kb fragment obtained by digestion of the bGH gene with PvuII was used as probe. Lanes 1–3, liver from control rabbits; lanes 4 and 5, liver from Tg1 and Tg2, respectively; lanes 6–8, kidney from control rabbits; lanes 9 and 10, kidney from Tg1 and Tg2, respectively.

The present study was performed with the two transgenic founder females, and nontransgenic female littermates were used as controls. At 3 months of age, the transgenic rabbits had high levels of serum bGH, whereas the hormone was not detected in serum from control rabbits ( Table 1). The transgenic rabbit that had integrated the highest number of copies of the transgene (Tg2) showed higher (about 4.5-fold) serum bGH concentration than Tg1. Moreover, higher serum IGF-I levels are observed in the transgenic rabbits compared to controls ( Table 1). This increase in IGF-I was related to the levels of circulating bGH, since Tg2 showed higher levels (about threefold) of IGF-I than Tg1. These results indicate that the transgene was expressed in the transgenic rabbits and that the hormone was secreted to the blood. When these animals were killed (Tg2 at 5 months of age and Tg1 at 10 months of age), the tissue distribution of the expression of the transgene was determined. Bovine GH mRNA was detected in the liver and kidney of transgenic rabbits ( Fig. 1C), indicating that the fragment of the PEPCK promoter used was able to direct the expression of the bGH gene to the tissues where the endogenous PEPCK gene is expressed. These results are consistent with those reported in transgenic mice expressing the same chimeric gene (16). As expected, no expression of the transgene was noted in skeletal muscle or pancreas of transgenic rabbits (data not shown).


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  		Table 1. Serum levels of bGH and IGF-I1fna

Expression of GH in transgenic mice leads to a large increase in growth rate and body size (14–16). To study the effects of overexpression of GH on the somatic growth of rabbits, the body weight of transgenic and control animals was determined at 1 month of age (weaning) and at 2 months of age (end of the growing period, when rabbits are usually commercially slaughtered). As indicated in Table 2, Tg1 and Tg2 both showed lower (about 25%) body weight at weaning than control rabbits. However, this reduction was not maintained at 2 months of age. At that time, Tg1 showed similar body weight, whereas that of Tg2 was slightly lower compared to control rabbits of the same age. Thus, transgenic rabbits overexpressing GH did not show the giant phenotype described in transgenic mice, which present increased overall growth for the various skeletal elements but maintain proportions similar to adult control mice (22). On the other hand, at 8–10 months of age, transgenic pigs expressing human or bovine GH do not grow to a larger body size, nor are femur, tibia, or humerus longer in transgenic pigs expressing bGH than in sibling controls (17). In contrast, enlargement of the head was observed in adult transgenic rabbits ( Fig. 2), a feature also found in acromegalic patients, which present hyperostosis frontalis and enlargement of hands and feet (13, 23). Furthermore, limbs from transgenic rabbits were longer than those from controls ( Fig. 2), which gives a `cat-like' appearance to these rabbits.


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  		Table 2. Body weight (g)2fna



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  		Figure 2. A control (A) and transgenic Tg1 (B) rabbits at 8 months of age. Note the enlargement of the head and the longer limbs of the transgenic rabbit.

Transgenic rabbits expressing the PEPCK/bGH chimeric gene were lean, having much less body fat than controls. Lack of perirenal fat can clearly be observed in Fig. 3. Similarly, transgenic pigs overexpressing GH are leaner than controls, and a marked reduction (40–60%) in back fat has been reported (17, 24). A reduction of fat mass has been also described in patients with acromegaly (25).



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  		Figure 3. Macroscopic observation of perirenal fat from a control (A) and transgenic Tg1 (B) rabbit. Note the lack of perirenal fat in the transgenic rabbit.

Long-term exposure to GH has been shown to lead to hyperinsulinemia and insulin resistance in transgenic mice (6) and pigs (24). Serum parameters were measured to study whether chronic exposure to GH led to metabolic alterations in transgenic rabbits. At 3 months of age, Tg1 showed mildly elevated (about 50%) serum insulin levels whereas Tg2 was markedly hyperinsulinemic with fivefold higher insulin concentration ( Table 3). This rabbit also developed hyperglycemia at this age, whereas Tg1 was normoglycemic. In addition, a marked elevation of serum triglycerides was observed in Tg2 at that age. These results indicate that Tg2 developed diabetes mellitus early in life, probably as a result of the high GH levels. Since Tg1 had lower levels of GH than Tg2, metabolic parameters were measured when Tg1 was aged 10 months to determine whether or not long-term exposure to bGH might lead to insulin resistance. At this age, Tg1 was hyperinsulinemic (about 2.5-fold increase) and hyperglycemic, and showed an increase in triglycerides and cholesterol, indicating that this rabbit had also developed insulin resistance ( Table 3). These results suggest that chronic exposure to moderate hyper-GH might also lead to insulin resistance and diabetes mellitus. These metabolic alterations observed in the transgenic rabbits were also consistent with the pattern of insulin resistance and frequent glucose intolerance observed in acromegalic patients (11, 12).


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  		Table 3. Serum parameters3fna

To study histopathological alterations resulting from chronic exposure to both GH and hyperinsulinemia, Tg2 and Tg1 were killed at 5 and 10 months of age, respectively. The liver of transgenic rabbits showed a marked sclerosis in the periportal area and degeneration of hepatocytes around the central veins ( Fig. 4A, B). However, the size of hepatocytes from transgenic rabbits was similar to that of control rabbits. In contrast, hepatocytes from transgenic mice overexpressing GH are enlarged (26). Analysis of kidney sections from the transgenic rabbits revealed that many glomeruli showed glomerulosclerosis and a thicker capsule of Bowman. Moreover, a generalized dilatation of Bowman's space was detected and a large proportion of the glomeruli showed atrophy ( Fig. 4C, D). Furthermore, a massive dilatation of renal tubules was observed ( Fig. 4C, D). Between the renal tubules, several zones of focal sclerosis were also found in the kidney of transgenic animals (data not shown). Histopathological analysis of skeletal muscle showed sclerotic lesions, some hypertrophic fibers, fiber degeneration, and calcifications ( Fig. 4E, F). These are well-defined lesions of acromegaly (27, 28), with aproximately 40% of patients presenting overt myopathy and mild muscle weakness and atrophy (27, 28).



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  		Figure 4. Histopathological analysis of liver (A, B) (x100), kidney (C, D) (x200), and skeletal muscle (E, F) (x100) from control (A, C, E) and transgenic Tg1 (B, D, F) rabbits. Sections were stained as indicated in Materials and Methods. In the transgenic rabbit, marked sclerosis of the liver, atrophy of kidney glomeruli, massive dilatation of renal tubules, and sclerotic lesions in skeletal muscle can be observed.

Generalized sclerotic lesions and skeletal abnormalities of acromegaly are irreversible. Cardiac and kidney complications are major causes of death in diabetic and acromegalic patients (13, 23). Furthermore, a rise in GH may also contribute to the pathology associated to diabetes (29). In addition, GH is currently used in chronic treatment of children retarded in growth, injured adults, and AIDS patients (30, 31). All this increases the need for animal models with which to study the long-term effects of GH. Transgenic mice expressing GH have been used as such a model (6, 26) because they develop glomerulosclerosis, hepatocellularmegaly, and myocardium sclerosis (26). However, these mice show a giant phenotype that is not observed in acromegaly (14–16). Transgenic pigs expressing GH do not grow more than controls, and although they develop severe alterations characteristic of their species, such as arthritis and stomach ulcers (17, 18), they do not show acromegalic sclerotic alterations (19). In contrast, transgenic rabbits expressing GH developed human-like acromegalic lesions (skeletal abnormalities and generalized-sclerotic lesions). However, it was difficult to obtain enough transgenic rabbits to perform the studies. This might be overcome in the future by generating more founder animals and by the use of low expressor, nonsterile transgenic males or ovary transplantation, as performed in transgenic mice expressing human GH (32).

Transgenic rabbits may therefore be a good model of acromegaly as they develop skeletal, metabolic, and histopathological alterations. The degree of insulin resistance in these animals was consistent with the levels of GH, as observed in acromegaly (12, 13). Our results also suggest that chronic exposure to GH might lead to such alterations and should be carefully considered when human patients are under long-term GH therapy. In addition, transgenic rabbits expressing GH might be useful to assay new therapies, such as GH antagonists, for acromegalic patients that are not eligible for surgery, as well as for other secondary alterations of prolonged exposure to GH.


   ACKNOWLEDGMENTS
 
We thank Dr. R. W. Hanson for the PEPCK/bGH chimeric gene; Drs. K. Burki and M. Massoud for transgenic rabbit technology instructions; Drs. J. Tarragó and O. Rafel (IRTA, Generalitat Catalunya) for providing the rabbits used in this study; R. Casamitjana for hormone measurements; and C. H. Ros, M. Moya, and A. Vilalta for technical assistance. C.C. and G.S. were recipients of predoctoral fellowships from the Ministry of Education, Spain, and Direcció General de Recerca, Generalitat de Catalunya, respectively. J.V. was the recipient of a postdoctoral fellowship from Direcció General de Recerca (1995SGR00518). This work was supported by grants from Fondo de Investigaciones Sanitarias (FISss 95/1758) and from Caixa de Barcelona, Spain.


   FOOTNOTES
 
1 Correspondence: E-mail: fatima.bosch{at}blues.uab.es

2 Abbreviations: GH, growth hormone; IGF, insulin-like growth factor; PEPCK, P-enolpyruvate carboxykinase; bGH, bovine growth hormone; RIA, radioimmunoassay; Tg1, transgenic rabbit 1.

Received for publication April 3, 1998.
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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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