Departments of 1Physiology, 2Pharmacology and Clinical Pharmacology, and 3Pediatrics, University of Turku, 20520 Turku, Finland; and 4Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Submitted 19 August 2002 ; accepted in final form 21 May 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
transgenic mice; polycystic ovarian syndrome; corticosterone; thermogenesis; hyperphagia
In the present study, we have used transgenic (TG) mice (bLH-CTP) with overproduction of LH (24). The transgene consists of the bovine
-subunit promoter fused with bovine LH
gene, which is extended with the coding sequence for the 24-amino acid COOH-terminal peptide of the human chorionic gonadotropin (hCG)
-subunit. Adult male mice harboring the same transgene do not have constitutively elevated LH secretion as females, probably because of different regulation of the
-promoter between the two sexes (24). However, in females, expression of the transgene leads to highly elevated levels of LH. This results in increased ovarian testosterone and estradiol (E2) secretion and extensive pathological changes in the ovaries, such as enlargement, formation of multiple follicular cysts, strain-dependent granulosa cell tumors, luteomas, precocious puberty, and infertility (11, 24, 25). This mouse line thus provides a useful model of functional ovarian hyperandrogenism, associated with cystic alterations. In addition, we have recently shown that the high LH levels in the bLH
-CTP females induce LH receptor expression in the adrenal cortex and stimulate corticosterone production, hence causing a phenotype reminiscent of Cushing's syndrome (12). LH-stimulated ovarian estrogen production resulted in increased prolactin production, which in turn could synergize with LH in the induction of adrenal LH receptor expression and LH responsiveness. There is ample evidence in rodents about upregulation of LH receptor expression by prolactin (6, 10). In humans, chronically elevated cortisol concentration can increase body fat, as seen in Cushing's syndrome (19, 23). The exact role of glucocorticoids in idiopathic obesity is poorly understood. Overall, the current knowledge suggests that, although circulating cortisol concentrations are normal in patients with idiopathic obesity, the secretion rates may be higher, paticularly in patients with visceral adiposity (7, 18). In addition, in some, but not all cases, mutations causing dysfunctional glucocorticoid receptor and increased cortisol concentration are associated with obesity (14, 26).
The objective of this study was to characterize in more detail the obesity encountered in the bLH-CTP mouse model. Herein, we describe its development and relationship with the mechanisms regulating body weight, appetite, and food intake.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Measurement of serum concentrations of corticosterone, insulin, leptin, glucose, cholesterol, triglycerides, and high density lipoprotein. Serum corticosterone was measured after dichloromethane extraction by RIA, using a polyclonal rabbit antiserum against corticosterone (kindly donated by Dr. R. Hampl, University of Prague, Czech Republic) and [1,2,6,7-3H]corticosterone (Amersham, Bucks, UK) as tracer (12). Serum insulin and leptin concentrations were measured by an RIA kit according to the manufacturer's protocols (Rat Insulin and Leptin RIA kits, Linco, St. Charles, MO). Serum glucose, cholesterol, triglycerides, and high density lipoprotein (HDL) were measured in the Clinical Laboratory of Turku University Hospital using a Hitachi 917 Automatic Analyzer (Hitachi, Tokyo, Japan). Except for the case of the insulin tolerance tests, venous blood glucose was measured using a Medisense glucose meter (Medisense, Bedford, MA) with glucose electrodes.
GDP binding to the mitochondrial fraction of brown adipose tissue. Binding of [3H]GDP to the brown adipose tissue (BAT) was measured using the method by Nicholls (20) with modifications described earlier (16). Fresh BAT was minced, diluted in 250 mM ice-cold sucrose buffer, and homogenized with a Potter S homogenizer (Braun, Melsungen, Germany). The mitochondrial fraction was isolated by differential centrifugation (28). [3H]GDP binding was determined by incubating the purified mitochondria in a medium containing 100 mM sucrose, 20 mM TES, 1 mM EDTA, 10 mM choline chloride, 2 M rotenone, 0.125 mCi/l [14C]sucrose, and 0.53 Ci/mmol of 10 µM [3H]GDP at room temperature for 10 min. Nonspecific binding was assessed in the presence of excess unlabeled GDP (1 mM). After incubation, the reaction was terminated by filtering the mixture through glass fiber filters (Thomas Scientific, Swedesboro, NJ) with a Brandel Cell Harvester (Biomedical Research Devices, Gaithersburg, MD). The GDP binding was assessed by measuring the radioactivity bound to the mitochondria by immersing the filters in a scintillation cocktail (Optic Phase "High Safe" II; FSA Laboratory Supplies, Loughborough, UK) and measuring the radioactivity in a liquid scintillation counter (LBK Wallac 1219, Turku, Finland). The amount of bound [3H]GDP, corrected by the amount of [14C]sucrose, represented the GDP binding. The protein content of the mitochondrial suspensions was assayed as described previously (22). The results are expressed as binding of GDP per milligram mitochondrial protein.
Northern hybridization. Leptin mRNA was determined in the retroperitoneal white adipose tissue (WAT) using Northern hybridization. Total RNA was isolated using the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method, as previously described (4). Total RNA (20 µg) was resolved on 1% denaturing agarose gels and transferred to Hybond-N nylon membranes (RPN 303 N; Amersham, Aylesbury, UK). The membranes were prehybridized for at least 4 h at 37°C in hybridization solution containing 50% deionized formamide, 5x saline-sodium citrate, 5x Denhardt's solution, 0.5% SDS, and 50 mg/l heat-denatured calf thymus DNA. A 25-mer oligonucleotide probe (5'-GGTCTGAGGCAGGGAGCAGCTCTTG-3'), labeled with [-32P]ATP (3,000 Ci/mmol; Amersham), specific for mouse leptin mRNA, was used in the hybridization. Hybridization and washing of the membranes were performed according to the instructions of the membrane manufacturer, and the hybridization results were visualized by autoradiography using Kodak X-AR 5 film (Eastman Kodak, Rochester, NY).
Measurement of body temperature. Body temperature of the bLH-CTP mice and their non-TG-littermates was measured using a rectal probe equipped with a digital laboratory thermometer (Model BAT-12; Physitemp, Clifton, NJ). Before cold exposure, the body temperature was measured at room temperature and then at +4°C after 0.5, 2, and 22 h of exposure. The temperature was measured two to four times from each mouse at each time point.
Statistical analysis. The Statview program (version 5.01 for Windows; Abacus, Gary, NC) was used for ANOVA, followed by Fisher's Protected LSD post hoc tests. In all statistical tests, the difference was considered significant at P < 0.05. The data are presented as means ± SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Obesity of bLH-CTP mice is associated with increased food consumption, lower thermogenesis of BAT, increased WAT leptin expression, and increased glucocorticoid levels. To evaluate the reasons for obesity of the bLH
-CTP mice, we first measured their food consumption and BAT thermogenesis. As shown in Table 2, the absolute food consumption of the female bLH
-CTP mice was increased significantly, but, as expected, this was not so in the TG males. BAT thermogenic activity, as measured by a mithochondrial GDP-binding assay, was 34% reduced in the bLH
-CTP females (n = 11) compared with non-TG controls (n = 4; 230 ± 13 vs. 348 ± 45 pmol/mg mitochondrial protein, respectively, P < 0.05). In line with these results, we found that the uncoupling protein-1 mRNA expression in BAT was reduced in TG females compared with non-TG controls (data not shown). Because the BAT thermogenic activity was reduced in the bLH
-CTP female mice, we evaluated their adaptation to cold, but no difference in body temperature was found after 0.5 h, 2 h, or overnight adaptation to 4°C (data not shown).
|
We also found that the leptin mRNA expression in WAT was about twofold increased in the obese bLH-CTP females compared with non-TG littermates (Fig. 3). In addition, the serum corticosterone levels in female bLH
-CTP mice were elevated significantly compared with control littermates (492 ± 186 vs. 168 ± 110 µg/l, respectively, P < 0.05), as shown also before with this model (12). In male mice, there were no differences in corticosterone levels of the TG and control groups. As expected, the increased expression of WAT leptin mRNA resulted in significantly elevated levels of serum leptin (Fig. 4F) in transgenic females compared with controls. Serum glucose, in either randomly fed or overnight fasted 3- to 4-mo-old bLH
-CTP females, was slightly but not significantly increased compared with non-TG controls (Fig. 4). The serum insulin levels of the bLH
-CTP females aged 4 mo and older were increased significantly compared with controls (Fig. 4B). However, in insulin tolerance tests with 0.75 U/kg body wt insulin, no significant differences were found at either 3 or 56 mo of age between the bLH
-CTP females mice and controls (Fig. 4, C and D). Serum cholesterol levels of the bLH
-CTP mice were significantly lower than in control mice (Fig. 4E). The majority of cholesterol was of the HDL type in both groups (Fig. 4). There were no significant differences in serum triglyceride levels.
|
|
Gonadectomy of the bLH-CTP females reverses their LH-associated weight gain, hyperphagia, and increase in serum corticosterone. To characterize further how the abnormal ovarian function of the bLH
-CTP mice affects their weight gain, and to analyze whether the high level of LH alone could be the causative factor, we gonadectomized groups of mice at the age of 3 wk and followed their subsequent growth rates. The data indicated that gonadectomy totally abolished the differences in body weight gain between the bLH
-CTP females and controls. The weights at the age of 4 mo were 22.8 ± 1.0 g (n = 4) and 23.2 ± 0.9g(n = 4), respectively (Fig. 5). The weights of the gonadectomized bLH
-CTP females did not differ significantly from those of intact or gonadectomized non-TG mice. Furthermore, after gonadectomy, the differences in food consumption disappeared between the TG and non-TG female mice (data not shown). However, the gonadectomized non-TG males had at the age of 4 mo weights (25.1 ± 0.9 g, n = 5) that were significantly lower than in nongonadectomized male controls (31.7 ± 0.6 g, n = 4).
|
To specifically evaluate the role of DHT and/or E2 in hypephagia and weight gain, ovariectomized bLH-CTP female and control mice were treated with DHT, E2, or with their combination between weeks 315 of life. The treatment of the ovariectomized bLH
-CTP female mice with DHT increased their body weights to the same level as in the intact bLH
-CTP mice. In contrast, E2 significantly decreased both food consumption and body weights of the mice. In gonadectomized bLH
-CTP females, the treatment with DHT led to a significantly increased amount of WAT compared with the intact control, whereas E2 either alone or combined with DHT decreased it. In addition, E2 treatment also reduced the food consumption of gonadectomized bLH
-CTP female mice compared with nontreated controls. The replacement therapy of gonadectomized bLH
-CTP females with either DHT, E2, or both did not increase the corticosterone levels to those of the intact bLH
-CTP females (results not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Obesity of the LH-overexpressing females seemed to be principally the result of an increased amount of peritoneal WAT, as indicated by increased weight of the retroperitoneal fat pad. However, a part of the weight difference may have also been the result of the increased weight of the skeletal muscles caused by the anabolic effect of elevated serum androgen levels of these mice (24). As expected, also in gonadectomized control male mice, the body weights were reduced significantly compared with noncastrated controls. In addition to the anabolic effect, testosterone could be an important factor stimulating food intake of the bLH-CTP mice, as shown in other studies (4). In the present study, the treatment of the gonadectomized female mice with DHT alone did not significantly increase food intake, but it was able to prevent the hypophagic effect of the E2 treatment. The role of glucocorticoids in the hyperphagia of bLH
-CTP female mice was not specifically addressed in this study.
We have recently shown, and confirmed here, that the bLH-CTP female mice have high circulating corticosterone levels, express LH receptors in the adrenal cortex, and produce corticosterone in response to endogenous LH and exogenous hCG stimulation (12). Most probably, the LH-stimulated ovarian estrogen production causes increased prolactin production in these mice. Prolactin, in turn, has been reported to upregulate LH receptor expression in rodents (10). These data, together with the present study, show a close connection between the elevated secretion of LH and corticosterone, as well as the disturbances in energy metabolism leading to obesity. Corticosterone has also been shown to be an important regulator in other models of mouse obesity. Mice with mutations in the ob gene (ob/ob mice) encoding leptin present with severe obesity, are hyperphagic, and have decreased energy expenditure (21). These mice also have marked elevation of blood corticosterone levels, and their development of obesity has been shown to be dependent on corticosterone excess.
In addition to hormonal factors, nonshivering thermogenesis is an important regulator of energy metabolism in rodents. Thermogenesis is strongly activated when mice are exposed to cold or when they ingest an excess of calories. Several obese rodent models show impaired BAT thermogenesis, which contributes to the development of obesity (8). The present study showed that, in the bLH-CTP mice, BAT thermogenic activity was reduced despite increased food consumption. The reason for this remains unknown, but the reduced thermogenesis in the bLH
-CTP females is supported by previous results showing that corticosterone decreases thermogenesis and increases lipid storage in BAT (30).
Our finding that leptin mRNA expression in WAT and serum leptin concentrations was increased in bLH-CTP females is also supported by previous studies showing a correlation between body fat and leptin concentrations (3). Furthermore, glucocorticoids have been shown to stimulate expression of the ob gene (29) and thus to have a role in elevating serum leptin levels in the bLH
-CTP females. The estrogens, with approximately twofold increased levels in bLH
-CTP females, might also stimulate leptin expression and release as has been suggested by other studies (2). Androgens, in contrast, are reported to inhibit leptin expression (31). In summary, the main hormonal changes causing the obese phenotype in the bLH
-CTP females are most probably the elevated levels of androgens and glucocorticoids. An increase in leptin expression and serum levels probably also stems from the hormonal changes but could be secondary to the increased amount of adipose tissue. A close correlation between the degree of adiposity and serum leptin levels has been reported in many studies (16). Elevated serum leptin levels have been reported in patients with PCOS compared with women without this syndrome (1), but, compared with controls matched for body mass index, leptin levels did not differ significantly (17, 27).
Despite significantly increased serum insulin in the bLH-CTP female mice, compared with control mice, the insulin tolerance test used in this study did not demonstrate clear signs of insulin resistance in these transgenic mice. In human PCOS patients, however, both hyperinsulinemia and insulin resistance are common (9, 13).
In this study, the genetic background of the mice was also found to influence the extent of weight gain. Although this phenomenon was not addressed specifically, the finding is interesting, because the strain of the mice used has recently also been shown to contribute to other special phenotypic characteristics, such as ovarian tumorigenesis (11). Notwithstanding the strong element of environmental factors in obesity, genetics has also been shown to play a major role in humans (15) and mice (33). Further evaluation of the genes related to obesity in the bLH-CTP mice may lead to novel candidates for studying the genetic causes of weight control in humans.
In conclusion, our results demonstrate that mice having chronically elevated levels of circulating LH are obese, have increased food consumption, and have reduced thermogenic activity of BAT. The concomitant endocrine changes indicate increased ovarian estrogen and androgen production, increased pituitary prolactin secretion, and induction of LH-dependent overproduction of adrenal corticosterone. The role of ovaries in the phenotype is obvious because ovariectomy normalized the corticosterone levels and prevented the increased body weight and food consumption that occurred in intact bLH-CTP females. Androgen replacement therapy in the ovariectomized bLH
-CTP females led to similar weight gain and also increased WAT as in intact TG females. However, a part of the obesity is likely the result of the LH-dependent increase in adrenal glucocorticoid secretion, which in turn is dependent on stimulation of the effect of the ovarian estrogen-prolactin link. The latter hormone apparently stimulates LH receptor expression in adrenal glands of the bLH
-CTP females. It remains to be studied to what extent this intriguing TG mouse model will provide insight into such human diseases as, e.g., PCOS, a syndrome with similar hormonal and metabolic alterations.
![]() |
DISCLOSURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|