©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of ob Gene Expression by Corticosteroids Is Accompanied by Body Weight Loss and Reduced Food Intake (*)

Piet De Vos , Régis Saladin , Johan Auwerx , Bart Staels (§)

From the (1)Laboratoire de Biologie des Régulations des Eucaryotes, Département d'Athérosclérose, Institut Pasteur, 59019 Lille Cédex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Genetic studies in mice have identified the ob gene product as a potential signaling factor regulating body weight homeostasis and energy balance. It is suggested that modulation of ob gene expression results in changes in body weight and food intake. Glucocorticoids are shown to have important metabolic effects and to modulate food intake and body weight. In order to test the hypothesis that these metabolic effects of glucocorticoids are linked to changes in the expression of the ob gene, ob mRNA levels were evaluated in rats treated with different glucocorticosteroids at catabolic doses and correlated to the kinetics of changes in body weight gain and food intake. Results from time course experiments demonstrate that adipose tissue ob gene expression is rapidly induced by glucocorticosteroids. This induction is followed by a concordant decrease in body weight gain and food consumption. These data suggest that the catabolic effects of corticosteroids on body weight mass and food intake might be mediated by changes in ob expression. Modulation of ob expression may therefore constitute a mechanism through which hormonal, pharmacological, or other factors control body weight homeostasis.


INTRODUCTION

Obesity, which can be defined as a body weight more than 20% in excess of the ideal body weight, is a major health problem in Western societies, since it is associated with an increased risk for cardiovascular disease, diabetes, and an increased mortality rate(1) . Obesity is the result of a positive energy balance, as a consequence of an increased ratio of caloric intake to energy expenditure. The molecular factors regulating food intake and body weight balance are incompletely understood. Five single-gene mutations resulting in obesity have been described in mice, implicating genetic factors in the etiology of obesity(2) . In the ob mouse a single gene mutation, obese, results in profound obesity, which is accompanied by diabetes(3) . Cross-circulation experiments have suggested that ob mice are deficient of a blood-borne factor regulating nutrient intake and energy metabolism(4) . Using positional cloning technologies, the mouse ob gene, and subsequently its human homologue, have been cloned recently(5) . Amino acid sequence analysis revealed a high degree of homology between mouse and human ob, indicative of an important regulatory function of this protein. The ob gene is exclusively expressed in white adipose tissue, suggesting that the protein can best be imagined as a fat-derived satiety factor. ob mice either have a non-sense mutation, resulting in the production of a non-functional gene product (C57Bl/6J ob/ob), or carry a genomic mutation resulting in the complete absence of ob mRNA (SM/Ckc-+-ob/ob) (5). ob mRNA levels are highly increased in C57Bl/6J ob/ob mice, suggesting that the level of expression of this gene signals the size of the adipose depot in an attempt to maintain body weight homeostasis.

The aim of the present work was to identify factors regulating ob gene expression and to evaluate whether changes in ob gene expression are correlated with changes in food intake and body weight. Since pharmacological doses of glucocorticoids have a strong catabolic action, the effects of high doses of glucocorticoids on the expression of the ob gene were studied and correlated to changes in body weight and food intake. The rat was chosen as a model, since body weight and adipose tissue mass keep increasing throughout its entire lifespan, thereby resembling the human situation of adult onset obesity. Furthermore, the relative short lifespan of this animal permits the study of factors affecting body weight homeostasis in a relatively short time period. The results from our studies demonstrate that administration of pharmacological doses of glucocorticoids induces adipose tissue ob gene expression. This induction is accompanied by reduced food intake and decreased body weight gain in these animals. These data indicate that modification of ob gene expression is subject to hormonal/pharmacological regulation, leading to the modulation of caloric intake and body mass gain.


MATERIALS AND METHODS

Animals and Treatments

Eighty-day-old male rats received once daily subcutaneous injections with the indicated corticosteroids at a dose and for the period of time indicated. Control animals received saline only. Rats were group-housed and accustomed to a 12:12 h day-night illumination cycle. Animals were allowed free access to standard rat chow. Body weight (per animal) and food consumption (per treatment group) were measured at regular intervals throughout the experiment. At the end of the experiment, animals were killed between 9 and 10 a.m. by exsanguination while under ether anesthesia. Epididymal fat pads were removed immediately and frozen in liquid N.

RNA Analysis

Total cellular RNA was prepared by the acid guanidinium thiocyanate/phenol-chloroform method(6) . Northern and dot blot hybridizations of total cellular RNA were performed as described previously(7) . A mouse ob cDNA fragment spanning nucleotides +50 to +659 was cloned from adipose tissue by reverse transcription and polymerase chain reaction amplification (sense primer: 5`-CCA AGA AGA GGG ATC CCT GCT CCA GCA GC-3`; antisense primer: 5`-CCC TCT ACA TGA TTC TTG GGT ACC TGG TGG CC-3`)(5) . The resulting polymerase chain reaction fragment was cloned into pBluescript KS, and sequence analysis revealed complete identity to the reported mouse ob cDNA sequence(5) . A -actin cDNA clone was used as a control probe(8) . All probes were labeled by random primers (Boehringer Mannheim). Filters were hybridized to 1.5 10 cpm/ml of each probe as described(7) . They were washed once in 0.5 SSC and 0.1% SDS for 10 min at room temperature and twice for 30 min at 65 °C and subsequently exposed to x-ray film (X-Omat AR, Kodak). Autoradiograms were analyzed by quantitative scanning densitometry (Bio-Rad GS670 densitometer) as described(7) .


RESULTS

To study the effects of pharmacological doses of glucocorticoids on body weight, food intake, and ob gene expression, 80-day-old male rats were treated once daily during 20 days with 100 µg of hydrocortisone/g of body weight Sham-treated control rats exhibited a significant, steady gain in body weight throughout the treatment period, attaining approximately 110% of the initial body weight after 20 days (Fig. 1). Administration of hydrocortisone, however, completely prevented this gain in body weight and resulted in a slight decrease in body weight at the end of the treatment period (Fig. 1). This difference in body weight gain between control and treated animals became only gradually apparent; the first 2 days of treatment body weights did not differ significantly from controls, and only thereafter a gradually more pronounced difference was observed. Compared with untreated animals, hydrocortisone-injected animals consumed 10-15% less food throughout the entire treatment period (Fig. 2), indicating that a reduction of food intake may, at least in part, account for the lower gain in body weight after hydrocortisone treatment.


Figure 1: Treatment with hydrocortisone reduces gain in body weight. Adult male rats received for the indicated number of days once-daily subcutaneous injections of hydrocortisone (100 µg/g of body weight). Control animals received saline only. Body weights were recorded at regular intervals and are expressed as a percentage of pretreatment (day 0) body weight. Values represent the mean ± S.D. of 4 animals/group.




Figure 2: Treatment with hydrocortisone reduces food intake. Adult male rats (n = 4/group) were treated as described under Fig. 1. Total food consumption of each treatment group was measured at regular intervals and is expressed as a percentage of the food intake of a group of sham-treated controls.



Since the ob gene product has been suggested to be a factor modulating food intake and energy utilization, the regulation of adipose tissue ob mRNA expression by hydrocortisone was determined next. Treatment with hydrocortisone increased ob mRNA levels more than 2-fold, an effect that was already maximal after 2 days (Fig. 3). ob mRNA levels remained elevated throughout the entire treatment period. This induction was specific, since -actin mRNA levels remained constant throughout the entire treatment period (Fig. 3).


Figure 3: Kinetics of induction of adipose tissue ob mRNA by hydrocortisone. Adult male rats (n = 4/group) were treated as described under Fig. 1. Adipose tissue was isolated, RNA was extracted, and ob and -actin mRNA levels were measured as described under ``Materials and Methods.'' Values represent the mean ± S.D. of 4 animals and are expressed in relative absorbance units (R.A.U.) taking the pretreatment values as 100%. Statistically (analysis of variance, p < 0.05) significant differences are observed between values followed by different letters (-actin, not significant).



Corticosteroids are known to exert dual metabolic actions, reflected by a bitonic dose-response curve for body weight gain(9) . In order to evaluate the dose-dependent effects of hydrocortisone on body weight and ob gene expression adult rats were treated once daily during 20 days with three different doses of hydrocortisone (1, 10, or 100 µg/g of body weight), respectively, corresponding to doses at the top, the middle, and the bottom of the descending arm of the body weight gain-corticosteroid dose-response curve(9) . Treatment of adult rats for 20 days with different doses of hydrocortisone resulted in a dose-dependent reduction in body weight gain (Fig. 4A), which was accompanied by a dose-dependent induction of ob mRNA levels in adipose tissue (Fig. 4B).


Figure 4: Dose-dependent effects of hydrocortisone on body weight gain (A) and adipose tissue ob mRNA levels (B). Adult male rats (n = 4 animals/group) received during 20 days once-daily subcutaneous injections of hydrocortisone at the indicated doses. Control animals received saline only. PanelA, body weights were recorded at the beginning and end of the experiment and are expressed as a percentage of pretreatment (day 0) body weight. PanelB, at the end of the experiment adipose tissue was isolated, RNA extracted, and ob and -actin mRNA levels measured as described under ``Materials and Methods.'' Values are expressed in relative absorbance units (R.A.U.) taking the controls as 100%. Values represent the mean ± S.D. Statistically (analysis of variance, p < 0.05) significant differences are observed between values followed by different letters (-actin, not significant).



Finally, the effects of the synthetic glucocorticoids dexamethasone and triamcinolone, which are relatively pure type II corticosteroid receptor agonists and produce a more pronounced monotonic negative dose-response curve of body weight gain(9) , were analyzed and compared with hydrocortisone. Treatment of adult male rats during 4 days with triamcinolone or dexamethasone also resulted in a marked decrease in body weight, which was paralleled by a similar decrease in food consumption (Fig. 5A). Concomitantly, ob mRNA levels increased after all three glucocorticoids tested (Fig. 5B). Northern blot hybridization analysis indicated that the ob cDNA probe hybridized to an mRNA of approximately 4.5 kilobases, a size similar to mouse adipose tissue ob mRNA(5) . Furthermore, ob mRNA levels already increased 2.2-fold in rat adipose tissue within 24 h after one single injection of dexamethasone (Fig. 5C), thereby indicating that the induction of ob gene expression by corticosteroids is a very rapid event.


Figure 5: Influence of different corticosteroids on body weight, food consumption (A), and adipose tissue ob mRNA levels (B and C). Panels A and B, adult male rats (n = 3 animals/group) were treated for 4 days with vehicle (CON), hydrocortisone (HC, 100 µg/g of body weight/day), triamcinolone (TRIAM, 20 µg/g of body weight/day), or dexamethasone (DEXA, 3.7 µg/g of body weight/day). Body weights and food consumption were recorded at the end of the experiment and are expressed as a percentage of the controls (PanelA). Adipose tissue was isolated, RNA extracted, and ob and -actin mRNA levels measured as described under ``Materials and Methods'' (PanelB). Values are expressed in relative absorbance units (R.A.U.) taking the controls as 100%. Values represent the mean ± S.D. Statistically (analysis of variance, p < 0.05) significant differences are observed between values followed by different letters (-actin, not significant). PanelC, adult male rats (n = 3 animals/group) were sacrificed 24 h after a single injection of dexamethasone (DEXA; 3.7 µg/g of body weight) or vehicle (CON). Total RNA (10 µg) extracted from individual animals was pooled and subjected to electrophoresis, transferred to a nylon membrane, and hybridized consecutively to labeled ob (toppanel) or -actin (bottompanel) cDNA as described under ``Materials and Methods.'' The positions of the 18 and 28 S rRNA bands are indicated on the right of the toppanel.




DISCUSSION

Genetically obese ob mice display a marked increase in body and adipose tissue mass and a pronounced hyperphagia. The cloning of the mouse and human ob genes and the demonstration that the ob gene is exclusively expressed in white adipose tissue and markedly increased in C57Bl/6J ob/ob mice suggest that the level of expression of this gene correlates with the size of the adipose depot. The ob gene product may therefore function as part of a pathway regulating body fat mass and food intake. An increase in the ob signal may act directly or indirectly on the central nervous system to inhibit food intake and/or regulate energy expenditure as part of a homeostatic mechanism to maintain body weight balance. It is therefore conceivable that the level of ob expression is inversely correlated with food intake, energy expenditure, and the onset of obesity.

In an attempt to understand the regulation of the ob gene and the impact of its regulation on body weight and food consumption, we studied the effects of different corticosteroids at pharmacological doses, which provoke severe metabolic perturbations and a reduction in food consumption. Our results demonstrate that glucocorticoids induce ob expression in rat adipose tissue, whereas a gain in body weight and food intake decrease concomitantly. Although the existence of a correlation does not prove causality, several lines of evidence argue in support of a causal relationship between the induction of ob gene expression, on the one hand, and the decrease in food intake and body weight, on the other hand. First, the induction of ob gene expression is very rapid and nearly maximal within 24 h after a single injection of corticosteroids. By contrast, the changes in body weight follow much more gradually, the difference with sham-treated controls only becoming significant after 3 days of treatment. Taking into account that a 16-h overnight fast reduces the body weight of rats by approximately 7.5% (fed, 376 ± 12 g; fasted, 350 ± 10 g), it appears that the effects of corticosteroids on body weight changes are much more gradual and lag behind the induction of ob gene expression. Second, the induction of ob expression by corticosteroids is independent of food intake since it is observed regardless of whether animals are fed or fasted. Third, it is unlikely that the alterations in ob expression are secondary to the decrease in food intake and body weight, since ob mRNA levels are increased in hyperphagic C57Bl/6J ob/ob mice. Finally, in contrast to normal mice, genetically obese ob/ob mice are dramatically resistant to glucocorticoid-induced weight loss(10) , indicating that the presence of a functional ob gene product is required to transmit the glucocorticoid-induced weight loss. Therefore, it seems that the induction of ob expression after corticosteroid treatment precedes and probably provokes the observed alterations in food intake and body weight. In this respect it is interesting to note that plasma corticosterone levels are elevated in obese C57Bl/6J ob/ob mice(11, 12, 13) , which may, at least in part, explain the increase in ob mRNA levels observed in these mice(5) .

Corticosteroids seem to exert a dual metabolic action on gain in body weight and feeding efficiency, strictly depending on the dose used(9) . Administration of high doses of glucocorticoids, such as in this study, have a dramatic catabolic action, resulting in a marked decrease in food intake and body weight. In contrast, administration of lower doses of corticosteroids has an anabolic activity, which is accompanied by increased appetite in humans and stimulation of food intake in laboratory animals. However, in contrast to their catabolic effects, it is unlikely that the anabolic effects of glucocorticoids, observed after administration of low doses, are mediated through changes in ob gene expression. Indeed, although ob/ob mice do not express a functional ob gene product, adrenalectomy reduces food intake and normalizes energy balance(14, 15, 16) , whereas corticosteroid replacement therapy restores food intake in these adrenalectomized ob/ob mice(17) . This unequivocally proves that the anabolic action of steroid hormones is ob-independent.

The effects of corticosteroids on ob gene expression may be due to a direct action of these hormones on ob gene transcription. Alternatively, these hormones may have indirect effects. Administration of high doses of glucocorticoids may, for instance, influence the plasma concentrations of other hormones that regulate food intake, such as dehydroepiandrosterone or corticotropin-releasing hormone. Alternatively, high doses of glucocorticoids increase gluconeogenesis, predispose to diabetes, and may therefore increase plasma glucose concentrations. According to the glucostasis theory, decreased plasma glucose concentrations would be a signal, triggering food intake. Thus, glucocorticoids may act by increasing plasma glucose concentrations, which in its turn may induce ob gene expression resulting in a reduction of food consumption. In this case factors involved in glucose metabolism, such as glucose itself, glucagon, and insulin, would be expected to be important modulators of ob gene expression.

In conclusion, we have shown that in vivo glucocorticoid treatment induces ob gene expression. Our data suggest that modulation of ob expression is inversely correlated with alterations in food intake and body weight. Knowledge of the regulation of ob gene expression and factors implicated therein will therefore be important in the prevention and treatment of obesity.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: L.B.R.E., Département d'Athérosclérose, Institut Pasteur, 1, rue du Prof. Calmette, 59019 Lille Cédex, France. Tel.: 33-20-877388; Fax: 33-20-877360.


ACKNOWLEDGEMENTS

We thank D. Cayet for excellent technical assistance and acknowledge the interesting discussions with Drs. M. Briggs, R. Heyman, R. Mukkerjee, J. Rosen, and J.-C. Fruchart.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.