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Endocrinology Vol. 140, No. 8 3609-3614
Copyright © 1999 by The Endocrine Society

ARTICLES

The Human Growth Hormone-Releasing Hormone Transgenic Mouse as a Model of Modest Obesity: Differential Changes in Leptin Receptor (OBR) Gene Expression in the Anterior Pituitary and Hypothalamus after Fasting and OBR Localization in Somatotrophs1

Aihua Cai and James F. Hyde

Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: James F. Hyde, Ph.D., Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, 800 Rose Street (MN224), Lexington, Kentucky 40536-0084. E-mail: jfhyde00{at}pop.uky.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We reported previously an increase in leptin receptor (OBR) gene expression in the anterior pituitary of human GH-releasing hormone (hGHRH) transgenic mice. The primary goal of this study was to investigate the possible mechanisms regulating OBR expression in these mice. Compared with normal sibling controls, hGHRH transgenic mice had significantly greater amounts of abdominal fat, higher levels of leptin messenger RNA (mRNA), and a 2-fold increase in plasma leptin concentrations. Despite normal plasma glucose levels, hGHRH transgenic mice had 4.5-fold elevated levels of plasma insulin. Using a ribonuclease protection assay, we measured the mRNA levels of the OBR long form (OBRL) in the anterior pituitary and hypothalamus after 48 h of fasting. In the anterior pituitary, food deprivation induced dramatic increases in OBRL mRNA levels in both normal and transgenic mice. In contrast, in the hypothalamus, fasting resulted in a significant decrease in OBRL gene expression in normal mice, and no changes were detected in hGHRH transgenic mice. Using dual in situ hybridization, OBRL mRNA was detected in somatotrophs. Moreover, the number of OBRL-positive pituitary cells as well as the percentage of OBRL-positive cells that express GH mRNA were increased in transgenic mice. In conclusion, 1) the modest obesity in hGHRH transgenic mice is associated with increases in leptin synthesis and secretion as well as insulin secretion; 2) GH and/or GHRH as well as leptin and insulin may differentially contribute to the changes in OBRL gene expression in the anterior pituitary and the hypothalamus; 3) the response of OBRL gene expression in the hypothalamus to fasting is absent in the modestly obese hGHRH transgenic mice; and 4) somatotrophs are target cells for leptin, and the increase in OBRL gene expression in the pituitary of hGHRH transgenic mice is due at least in part to the increase in the number of cells expressing OBRL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RECENTLY cloned leptin receptor (OBR) is expressed in many tissues (1, 2). The dominant isoform that transduces the leptin signal is the OBR long form (OBRL), and it is predominantly expressed in the hypothalamus (2, 3, 4). Therefore, the hypothalamus is currently considered the central target site of leptin. We and others suggested that the anterior pituitary is an additional target site of leptin (5, 6, 7). Leptin acts through its functional receptor in the hypothalamus and the anterior pituitary to regulate energy balance and maintain metabolic homeostasis. Mice and humans (8, 9) with genetic mutations of leptin or OBR develop obesity (3, 10, 11, 12). However, additional studies show that obese animals or individuals do not necessarily lack leptin, but, instead, leptin levels are elevated, suggesting that leptin resistance also leads to the onset of obesity (13, 14, 15).

Human GH-releasing hormone (hGHRH) transgenic mice have visibly larger amounts of abdominal fat, suggesting that they may be obese. These mice have a fusion gene that includes the promoter/regulatory regions of the mouse metallothionein I gene and the coding regions of the human GHRH gene (16). Besides increased body weight, resulting from high levels of circulating GH, few metabolic characteristics of these mice have been studied. For example, it is not known whether the chronic elevation of GH and/or GHRH in these mice contributes to other hormonal changes, such as in leptin and insulin, which, in turn, may lead to the onset of obesity. In the present studies, we characterized some metabolic aspects of the hGHRH transgenic mouse to explore it as an animal model of modest obesity. We reported previously a differential expression of the OBRL gene in the hypothalamus and the anterior pituitary of hGHRH transgenic mice (5). hGHRH transgenic mice showed elevated OBRL gene expression in the anterior pituitary, and no changes were detected in the hypothalamus compared with that in normal controls (5). The metabolic characterization of these mice would provide valuable information for possible mechanisms underlying the differential expression of the OBRL gene.

Despite the importance of leptin in regulating food intake and energy balance, the regulation of OBRL expression is poorly understood. To begin to understand the regulation of OBRL by food intake, we investigated the changes in OBRL gene expression in the anterior pituitary and hypothalamus caused by fasting. In hGHRH transgenic mice, it is plausible that changes in plasma leptin and/or insulin, secondary to the elevation of GH and/or GHRH, contribute to the tissue-specific changes in OBRL gene expression. Food deprivation decreases plasma levels of insulin, leptin, as well as GH (17, 18). Although no reports show how fasting changes these hormones in hGHRH transgenic mice, we expected decreases in the plasma levels of leptin and insulin, but in contrast to normal mice, we expected the retention of high levels of GH in the transgenic mice. Therefore, by fasting normal and hGHRH transgenic mice we could ascertain whether 1) OBRL gene expression in the hypothalamus and that in the anterior pituitary are regulated by food intake, 2) the differential changes in OBRL gene expression in the tissues are due to GH and/or GHRH, and 3) whether leptin and insulin play roles in the regulation of OBRL gene expression.

To further understand the roles of leptin in the regulation of pituitary gland function, it is essential to localize the specific cell types in the anterior pituitary that express the OBRL gene. In the hypothalamus, OBR gene expression has been reported in neuropeptide Y (NPY), POMC, and other neurons (19, 20, 21, 22). To our knowledge, no studies have reported the cells expressing OBR messenger RNA (mRNA) in the anterior pituitary. Our data (5) as well as those of other investigators (23) suggest that somatotrophs are an important candidate. Therefore, in the present studies we performed dual in situ hybridization to colocalize OBRL and GH mRNAs in the anterior pituitary. By analyzing the population of OBRL-positive cells, especially the population of cells that coexpress OBRL and GH mRNAs, in both normal and hGHRH transgenic mice, we will 1) better understand the interactions between leptin and somatotrophs, and 2) elucidate potential reasons for the dramatic increase in OBRL gene expression in the anterior pituitary of hGHRH transgenic mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and procedures
Male hGHRH transgenic and normal sibling mice (4–6 months old) were maintained in a constant temperature environment and a 14-h light, 10-h dark cycle (lights on at 0400 h). Animals were housed individually to measure their daily food and water consumption. During the fasting study, one group of normal and hGHRH transgenic mice was allowed free access to food and water; food was removed from another group of animals at 0900 h, and animals were killed 2 days later between 0900–1100 h. Body weights were measured before and after fasting, and length of each animal from the nose to the base of tail was determined before death. Trunk blood was collected to measure plasma leptin, insulin, GH, and glucose concentrations. Glucose concentrations were measured using a blood glucose monitor (Accu-Chek III, model 766, Boehringer Mannheim, Indianapolis, IN). The hypothalamus and the anterior pituitary were collected as previously described (24). Abdominal fat was collected and weighed. Total RNA from the hypothalamus, anterior pituitary, and fat was extracted according to the method of Chomczynski and Sacchi (25). For dual in situ hybridization, anterior pituitaries were obtained from normal and hGHRH transgenic mice (n = 3/group), enzymatically dispersed into single cells, and fixed on microscope slides as previously described (26).

Northern blot analysis
Leptin messenger RNA (mRNA) levels in abdominal fat were quantified by Northern blot analysis using 10 µg total RNA (27). A 48-base oligonucleotide complementary to mouse leptin mRNA (+271 to +318; GenBank accession no. U18812) and a 26-base oligonucleotide complementary to human 28S ribosomal RNA (28) were end labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase.

RIA
GH (24), leptin, and insulin levels in plasma were measured by RIAs. Leptin and insulin concentrations were measured using specific RIA kits (Linco Research, Inc., St. Charles, MO). The limits of sensitivity for the mouse leptin and insulin assays were 0.2 and 0.1 ng/ml, respectively. The intra- and interassay coefficients of variation were less than 10%.

Ribonuclease protection assay
The mRNA levels of OBRL in the hypothalamus and the anterior pituitary were measured by a ribonuclease protection assay as previously described (5). The complementary DNA (cDNA) template, a gift from ZymoGenetics, Inc. (Seattle, WA), was specific to the cytoplasmic domain of the mouse OBRL (+2469 to +3579; GenBank accession no. U46135). We verified its sequences by dideoxy chain termination sequencing (Sequenase, version 2.0, U.S. Biochemical Corp., Cleveland, OH) and linearized the cDNA with HincII. The OBRL complementary RNA (cRNA) was transcribed in vitro with 32P-labeled {alpha}-UTP. The protected product of the ribonuclease protection assay was 270 bp. ß-Actin was used as an internal control as previously described (5). Data are presented as the ratios of OB-RL/ß-actin per 10 µg RNA (mean ± SE; n = 5–9 mice/group).

Dual in situ hybridization
The colocalization of OBRL and GH mRNAs in the anterior pituitary was measured by dual in situ hybridization as previously described with minor modifications (26). OBRL and GH antisense cRNAs were labeled using digoxigenin- and 35S-labeled {alpha}-UTP, respectively, with a specific activity of 4.6 x 108 dpm/µg for the GH cRNA. Signals of digoxigenin-labeled OBRL mRNA were detected in chromagen solution, and then the slides were dipped in 1:1 diluted Ilford photographic emulsion (Polysciences, Inc., Warrington, PA). Signals for 35S-labeled GH mRNA were detected after 14 days of exposure at 4 C.

The numbers of somatotrophs, OBRL-positive pituitary cells, and cells coexpressing OBRL/GH were counted on the same slides probed with OBRL and GH cRNAs. Cells were counted from at least 20 different representative areas or approximately 2000 pituitary cells on each slide. Data are presented as the mean ± SE (n = 3) of the percentage of specific cell types in the anterior pituitary cell population.

Data analysis
Student’s t tests were performed to compare the body weight, body weight/nose-tail lengths, daily food intake, water consumption, abdominal fat weights, glucose concentrations, as well as different cell populations in the anterior pituitary. Two-way ANOVA was used to analyze leptin mRNA levels; plasma leptin, insulin, and GH concentrations; as well as OBRL mRNA levels. If a significant interaction between group (normal vs. hGHRH transgenic mice) and treatment (fed vs. fasted) was detected (P < 0.05), post-hoc comparisons were made using Student-Newman-Keuls tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the hGHRH transgenic mouse as a model of modest obesity
Compared with normal sibling controls, hGHRH transgenic mice had significantly greater body weight, consumed significantly (P < 0.01) greater amounts of food and water daily, and had higher body weight/nose-tail length ratios (Table 1Go). Moreover, their abdominal fat contents (milligrams of fat per g BW) were significantly (P < 0.01) greater than those of normal control mice (Table 1Go). To determine whether hGHRH transgenic mice were diabetic, we measured plasma insulin and glucose concentrations. Despite normal glucose levels (157.00 ± 7.07 vs. 158.25 ± 9.21 mg/dl in control and transgenic mice, respectively; P = 0.92), hGHRH transgenic mice had a 4.5-fold increase in insulin concentrations compared with normal controls (Fig. 1CGo). Furthermore, we found a 2-fold increase in plasma leptin concentrations in hGHRH transgenic mice, and the mRNA levels of leptin in abdominal fat were increased 1.7-fold concomitantly (Fig. 1Go, A and B). As reported previously (24), hGHRH transgenic mice had much higher levels of circulating GH than normal mice (Fig. 1DGo). A 48-h fast resulted in a significant decrease (P < 0.05) in the body weights of both normal (28.5 ± 0.54 vs. 23.95 ± 0.73 g) and hGHRH transgenic mice (39.7 ± 0.98 vs. 32.9 ± 1.12 g). Leptin mRNA in abdominal fat and plasma leptin levels as well as plasma insulin concentrations were also decreased significantly in both normal and hGHRH transgenic mice after fasting (Fig. 1Go, A–C). Plasma GH concentrations were decreased (P < 0.05) in normal mice after fasting, but those in hGHRH transgenic mice were increased significantly (P < 0.05; Fig. 1DGo).


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  		Table 1. Body weight, food and water consumption, and body weight/nose-tail length in control and transgenic mice

 


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  		Figure 1. Effects of fasting on leptin mRNA levels in abdominal fat (A) and plasma leptin (B), insulin (C), and GH (D) concentrations in normal (control; open bars) and hGHRH transgenic (hatched bars) mice. Data are presented as the mean ± SE (n = 6–10 animals/group·treatment). *, Significance (P < 0.05) compared with control fed mice; **, significance (P < 0.05) compared with transgenic fed mice.

 
Tissue-specific regulation of OBRL gene expression in the anterior pituitary and hypothalamus by fasting
In the anterior pituitary, hGHRH transgenic fed mice expressed a significantly higher level of OBRL mRNA compared with control fed mice as reported previously (5). Forty-eight hours of food deprivation resulted in significant increases in OBRL gene expression in both normal and hGHRH transgenic mice (Fig. 2AGo). Moreover, the increase in OBRL gene expression in hGHRH transgenic compared with control mice was still evident after food deprivation (Fig. 2AGo). In contrast to the anterior pituitary, 48 h of food deprivation resulted in a significant (P < 0.05) decrease in OBRL gene expression in the hypothalamus in normal control mice (Fig. 2BGo). In hGHRH transgenic mice, however, the levels of OBRL mRNA in the hypothalamus after 48 h of fasting remained unchanged compared with those in control fed and transgenic fed mice.



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  		Figure 2. Tissue-specific expression of OBRL mRNA in the anterior pituitary (A) and hypothalamus (B) of normal (control; open bars) and hGHRH transgenic (hatched bars) mice by fasting. Data are presented as the mean ± SE (n = 5–9 animals/group·treatment). A, Two-way ANOVA revealed no interaction (P = 0.11) between group and treatment, but significant main effects of group (P < 0.001) and treatment (P < 0.002) were evident. B, Two-way ANOVA revealed a significant interaction (P < 0.04) between group and treatment, and significant main effects of both group (P < 0.03) and treatment (P < 0.002).

 
Colocalization of OBRL and GH mRNAs in the anterior pituitary
Dual in situ hybridization revealed the colocalization of GH with OBRL mRNAs in both normal and hGHRH transgenic mice (Fig. 3AGo). In normal controls, 6.4% of cells in the anterior pituitary were OBRL positive, and 36.1% of them were somatotrophs (Fig. 3Go, B and C). In hGHRH transgenic mice, the number of OBRL-positive cells was increased to 20.1%, and 56.4% of the OBRL-positive cells were somatotrophs (Fig. 3Go, B and C).



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  		Figure 3. A representative photomicrograph of colocalization of OBRL and GH mRNAs in dispersed anterior pituitary cells from a hGHRH transgenic mouse using dual in situ hybridization. A, The large arrow points to a cell coexpressing GH and OBRL mRNAs, the small arrow points to a cell that expresses OBRL mRNA only, and the arrowhead points to a cell that expresses neither OBRL nor GH mRNA. B, Distribution of OBRL-positive (OBR+) cells in the total population of the anterior pituitary. C, Percentage of GH-positive cells in the population of OBRL-positive cells (GH+/OBR). Data from B and C are presented as the mean ± SE (n = 3 animals/group). *, Significance (P < 0.05) compared with normal controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
hGHRH transgenic mice are giants as a result of constant exposure to high circulating hGHRH and GH throughout their lifetime (16). GH is known to stimulate skeletal and muscle growth and promote lipolysis. Therefore, it is striking that hGHRH transgenic mice develop modest obesity. They displayed an increase in food intake, body weight, and abdominal fat contents compared with normal controls. Other investigators showed that chronic treatment with GH could produce hyperinsulinemia and insulin resistance (29). The diabetogenic activity of GH has been reported in both human and rodent studies (30, 31). The hGHRH transgenic mice have a 4.5-fold increase in insulin concentrations despite normal blood levels of glucose, indicating that these mice have developed hyperinsulinemia and insulin resistance. Similar changes of hyperinsulinemia and euglycemia are also reported in transgenic mice that overexpress bovine GH (32). Our data suggest that the constantly elevated GHRH/GH levels also lead or contribute to a steady state increase in leptin synthesis and secretion. Therefore, the modest obesity of hGHRH transgenic mice may be the result of insulin resistance combined with excessive leptin synthesis and secretion.

Consistent with reports from other investigators, fasting resulted in a decrease in plasma levels of GH as well as in leptin and insulin levels (17, 23). In contrast to normal controls, plasma concentrations of GH increased in hGHRH transgenic mice after 48 h of fasting. The transgene includes the promoter/regulatory fragment of the metallothionein I gene, which is regulated by heavy metals and glucocorticoids (33, 34). A long lasting, glucocorticoid-dependent, 10- to 20-fold increase in metallothionein I mRNA levels was noted after restraint stress in mice (35). The stress of fasting induces an increase in corticosterone (17), which we postulate probably activates the transgene and contributes to an even greater elevation of endogenous GH levels in the transgenic mice. Future studies will directly test this hypothesis.

Our present study suggests that GHRH and/or GH as well as leptin and insulin regulate, in a differential manner, OBRL gene expression in the anterior pituitary. We detected a similar pattern of higher OBRL gene expression in the anterior pituitary in both fed and fasted transgenic mice, suggesting that high levels of GH and/or GHRH maintain elevated OBRL gene expression in the anterior pituitary regardless of food intake. However, the increase in steady state levels of OBRL mRNA in fasted normal and transgenic animals suggests that factors other than GH also play a role in regulating OBRL gene expression in the anterior pituitary. OBRL gene expression in the pituitary after fasting was inversely correlated to plasma leptin and insulin concentrations in both normal and transgenic mice. Therefore, our data suggest that leptin as well as insulin may down-regulate OBRL gene expression in the anterior pituitary. As a result, the additive effects of elevated GH and decreased leptin and insulin levels in hGHRH transgenic fasted mice may have led to a much more dramatic increase in OBRL gene expression in the pituitary.

Data from dual in situ hybridization further enhanced our understanding of why OBRL gene expression in the anterior pituitary of hGHRH transgenic mice was elevated. This is the first study that quantitatively analyzed the population of OBRL-positive cells in the anterior pituitary. More than 30% of OBRL-positive cells were somatotrophs, suggesting that these cells are an important target for leptin, and that leptin may regulate metabolism by directly regulating GH synthesis and secretion (23). It is not surprising that cell types other than somatotrophs express OBRL, as leptin is also involved in reproductive functions (36, 37) and stress responses (6, 17). Thus, gonadotrophs and corticotrophs are likely candidates for targets of leptin. In hGHRH transgenic mice, we also detected a significant increase in the percentage of OBRL-positive cells as well as the percentage of cells coexpressing OBRL and GH mRNAs. It is likely that the increase in OBRL mRNA levels in hGHRH transgenic mice is due at least in part to the increase in the number of cells that express the OBRL gene. In addition, our data obtained using ribonuclease protection assays suggest that the levels of OBRL mRNA per pituitary cell (i.e. normalized with ß-actin mRNA) are increased.

The present studies indicated that the regulation of OBRL gene expression by fasting in the hypothalamus is independent of that in the anterior pituitary. Fasting for 48 h resulted in a significant decrease in OBRL mRNA levels in the hypothalamus of normal mice. Our data are in contrast to those reported by Bennett et al. (38). Using in situ hybridization, Bennett et al. detected an increase in OBRL gene expression in the thalamus after 72 h of fasting in female rats and no changes in hypothalamic nuclei, such as the arcuate and ventromedial nuclei. Differences in the animal model (female random cycling rats vs. male mice), techniques (in situ hybridization vs. ribonuclease protection assay), and tissues (specific hypothalamic nuclei vs. whole hypothalamic tissue) may contribute to the differences in the results. However, it is puzzling that in the same study it was reported that fasting induced a dramatic decrease in OBR mRNA, the combination of all isoforms, in arcuate and ventromedial nuclei, and OBRL is the dominant isoform of OBR in the hypothalamus (2, 3).

It is interesting that the decrease in OBRL gene expression in the hypothalamus of normal mice after fasting did not occur in modestly obese hGHRH transgenic mice. Moreover, the increase in circulating leptin and insulin did not lead to an elevation of OBRL gene expression in the hypothalamus of transgenic fed mice. The absence of changes in OBRL gene expression in the hypothalamus of hGHRH transgenic mice may be the consequence of leptin and insulin resistance and may also partially explain why these mice develop modest obesity. The failure of OBRL in the hypothalamus to respond to the peripheral changes in leptin may indicate an inability of hypothalamic target neurons to detect the changes in the energy milieu. This vicious cycle eventually may lead to an unbalanced metabolic status and the onset of obesity. Further studies in these mice are necessary to support this idea.

Fasting results in a variety of diverse changes involving many factors besides leptin, insulin, and GH. Ahima et al. showed that 48 h of fasting results in numerous endocrine changes, including alterations of thyroid hormones, ACTH, and corticosteroids (17). To our knowledge no studies have been performed to study the regulation of OBRL gene expression by these different hormones. In the hypothalamus, fasting increases NPY gene expression (39) and decreases POMC mRNA levels (40) in the arcuate nucleus. Moreover, GH feeds back on GH receptors localized on hypothalamic NPY neurons (41, 42). Thus, it is plausible that a combination of different hormonal changes leads to the differential changes in OBRL mRNA in the anterior pituitary and hypothalamus.

In summary, there is a tissue-specific regulation of OBRL gene expression in the anterior pituitary and the hypothalamus by fasting in both normal and modestly obese hGHRH transgenic mice, demonstrating the independent control of OBR in these two important endocrine tissues. In the anterior pituitary, somatotrophs are one of the direct targets of leptin. The increase in OBRL mRNA levels in the pituitary of hGHRH transgenic mice is due at least in part to the increase in the number of cells that express the OBRL gene.


   Acknowledgments
 
We thank ZymoGenetics, Inc., for providing us with the mouse leptin receptor cDNA, Dr. Kelly Mayo (Northwestern University) for supplying us with a founder hGHRH transgenic mouse, and Dr. A. F. Parlow (National Hormone and Pituitary Program, University of California-Los Angeles-Harbor Medical Center) for providing the reagents for the mouse GH RIA.


   Footnotes
 
1 This work was supported in part by NIH Grants DK-45981 (to J.F.H.) and HD-07436 (to A.C.) and the University of Kentucky Medical Center Research Fund. Preliminary results of this investigation were presented at the 28th Annual Meeting of the Society for Neuroscience, Los Angeles, California, 1998. Back

Received November 2, 1998.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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