©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Down-regulation of the Expression of the Obese Gene by an Antidiabetic Thiazolidinedione in Zucker Diabetic Fatty Rats and db/db Mice (*)

(Received for publication, August 1, 1995; and in revised form, January 30, 1996)

Bei Zhang (§) Michael P. Graziano (1)(¶)(**) Thomas W. Doebber (**) Mark D. Leibowitz (**) Sylvia White-Carrington Deborah M. Szalkowski Patricia J. Hey (1) Margaret Wu Catherine A. Cullinan Philip Bailey (2) Bettina Lollmann (3) Robert Frederich (3)(§§) Jeffrey S. Flier (3)(¶¶) Catherine D. Strader (1)(¶) Roy G. Smith

From the  (1)Departments of Molecular Endocrinology, Molecular Pharmacology and Biochemistry, and (2)Endocrine Pharmacology, Merck Research Laboratories, Rahway, New Jersey 07065 and the (3)Department of Endocrinology, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Obese (ob) is a recently identified gene involved in the regulation of energy balance in the mouse. We report here that AD-5075, a potent thiazolidinedione which lowered plasma glucose and triglyceride in Zucker diabetic fatty (ZDF) rats and db/db mice, decreased the expression of the ob gene in these animal models of obesity and non-insulin-dependent diabetes mellitus. The level of adipose ob mRNA in ZDF rats was 3-fold greater than that detected in the Zucker lean littermates. Chronic treatment with AD-5075 elicited a 67 and 70% reduction of ob mRNA in ZDF and control lean rats, respectively. Furthermore, the amount of adipose ob mRNA in db/db mice was 7 times higher than that detected in lean littermates. Treatment of db/db mice with AD-5075 resulted in a 78% reduction of the level of ob mRNA with parallel changes in circulating level of the ob gene product, leptin. The reduction of the ob mRNA in the Zucker lean rats was accompanied by significantly greater food intake and weight gain. However, in ZDF rats and db/db mice, there was profound increase in body weight without hyperphagia. The results demonstrate that the expression of the ob gene is up-regulated in these two rodent models of diabetes compared to their lean counterparts and that such overexpression is attenuated by treatment with an agent that improves insulin sensitivity and glucose homeostasis in vivo.


INTRODUCTION

Obesity is a predisposing factor for insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM) (^1)in both humans and animals(1) . Although the genetics of the obesity-NIDDM syndrome is poorly understood in humans, a number of loci associated with obesity have been mapped to different chromosomes in rodents(2) . Obese (ob) is a recently identified gene involved in the regulation of energy balance in the mouse(3) . The ob gene product, leptin(4) , expressed predominantly in white adipose tissue, is believed to be a secreted protein that acts as a satiety factor. Mice homozygous for a mutation in the ob gene are deficient in leptin, consume much more food than their lean counterparts, and develop severe obesity and characteristics of insulin resistance and diabetes. Administration of recombinant mouse leptin resulted in a significant reduction of food intake and body weight as well as a normalization of metabolic status in ob/ob mice(4, 5, 6) , consistent with the hypothesis that leptin is a key hormone in the control of energy intake and expenditure.

Since leptin plays a pivotal role in the regulation of adiposity and energy homeostasis, the level of its expression is likely to fluctuate under various physiological, nutritional, and disease conditions. Increased expression of ob mRNA has been reported in mice with deregulated body weights as results of mutations at the db locus(7) , lesions of the hypothalamus induced by gold thioglucose (7) or monosodium glutamate(8) , and transgenic ablation of brown adipose tissue(8) . Furthermore, the level of ob mRNA and the circulating level of leptin are altered in response to starvation and refeeding(8) . In addition, glucocorticoids induce the expression of the ob gene with a concomitant reduction of body weight and food intake in rats(9) . Thus, the expression of the ob gene is modulated by a variety of factors that influence adipose mass, including hormone levels, nutrients, and metabolic status in several rodent models.

The present study was initiated to gain insight into the regulation of ob gene expression by an antidiabetic thiazolidinedione in ZDF rats and db/db mice, two widely studied rodent models of NIDDM. Thiazolidinediones are a class of agents efficacious in lowering plasma glucose, reducing hyperinsulinemia, and correcting aberrant glucose metabolism in animal models of NIDDM (10, 11, 12) and in improving insulin sensitivity in cultured cells(13, 14) . Recently, members of this class of agents have been identified as high affinity ligands for the peroxisome proliferator-activated receptor (PPAR), a transcription factor that modulates the expression of various adipose-specific genes(15) . The results of the present study demonstrate that in addition to being an effective antidiabetic agent, a thiazolidinedione (AD-5075) down-regulates the expression of the ob gene in the two animal models that were studied. Therefore, ob gene expression is subject to modulation by a pharmacological agent that ameliorates insulin resistance and improves glucose homeostasis. The reduction of the level of ob gene expression is also accompanied by changes in body weight and energy intake.


EXPERIMENTAL PROCEDURES

Animals

Male C57BL/KsJ-db/db mice and lean littermates (+/?) were obtained from Jackson Laboratories. Male Zucker diabetic fatty (ZDF-fa/fa) rats and Zucker lean littermates (+/?) were from Genetic Models, Inc. The animals were 10-14 weeks old when dosing began. AD-5075 (AD, 5-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl]-2,4-thiazolidinedione) was administered via oral gavage for 4 weeks at doses of 2 and 5 mg/kg/day, for db/db mice and ZDF rats, respectively. The doses were chosen based on previous experiments in order to achieve near normalization of plasma glucose and triglyceride concentrations. All animals received milled rodent chow (Purina 5008) ad libitum and had free access to water. Food intake, body weight, plasma glucose, and triglyceride levels were monitored throughout the dosing period.

RNA Isolation and Analysis

-Total RNA was prepared from epididymal fat pads using the Ultraspec RNA isolation system (Biotecx). The RNA concentration was estimated by measuring absorbance at 260 nm. For Northern analyses, 10 µg of RNA was denatured in formamide/formaldehyde and electrophoresed in a formaldehyde-containing 1% agarose gels(16) . RNA was transferred to a Hybond-N membrane (Amersham) by capillary blotting. Prehybridization was performed at 42 °C for 1 h in 40-50% formamide in a solution containing 25 mM sodium phosphate (pH 7.4), 0.9 M sodium chloride, 50 mM sodium citrate, 0.1% each of gelatin, Ficoll, and polyvinylpyrollidone, 0.5% sodium dodecyl sulfate (SDS), and 100 µg/ml denatured salmon sperm DNA. Hybridization was carried out at 42 °C for 20 h in the same solution with P-labeled cDNA probes as described below. After washing the membranes under appropriately stringent conditions, the hybridization signals were analyzed with a PhosphorImager (Molecular Dynamics).

A cDNA probe for ob was obtained by cloning the entire coding region of ob using polymerase chain reaction based on the published sequence(3) . Briefly, total white adipose RNA was isolated from Swiss-Webster mice and first strand cDNA synthesized. Using polymerase chain reaction the coding region of the ob cDNA was isolated as 2 overlapping fragments using the following primer sets (5`-CAGTGAGCCCCAAGAAGAGG-3`, 5`-TCCAGGTCATTGGCTATCTG-3`, and 5`-ATTCCTGGGCTTCAGGGGATTCTGAGTTTC-3`, 5`-GCGTGTACCCACGGAGGAAC-3`). The resulting 380- and 626-base pair fragments were purified and used as templates in a subsequent polymerase chain reaction with primers 5`-AAGAATTCATGTTGCTGGAGACCCCTGTGTC-3` and 5`-AAGGATCCTCAGCATTCAGGGCTAACATC-3`. The final 501-base pair fragment was used as the ob cDNA probe. The cDNA probe for mouse adipocyte fatty acid-binding protein (FABP) (aP2) was obtained from Dr. David Bernlohr (University of Minnesota). The probes were labeled with the Oligolabeling Kit (Pharmacia), using [alpha-P]deoxy-CTP.

Immunoblotting of Plasma Leptin Protein

Polyclonal rabbit antibodies were raised against a C-terminal (RLQGSLQDILQQLDVSPEC) peptide of the predicted leptin protein(3) . The antibody was affinity purified using Sulfolink^R antibody purification kit (Pierce) following the manufacturers instruction. Western blots using this antibody detect a 16-kDa protein in the media of HEK-293 cells transfected with pCMV vector containing the coding region of the ob cDNA(8) . The specificity of the antibody was confirmed by the absence of the 16-kDa protein in the media of 293 cells transfected with the vector alone, its disappearance when the rabbit serum was preincubated with excess peptide antigen, and its absence from the serum of ob/ob mouse homozygous for the truncation mutation prior to the C-terminal peptide(8) . Six microliters of plasma from control and AD-5075-treated mice were separated by electrophoresis on 16% Tricine-SDS gels. The proteins were transferred to Immobilon PVDF membranes (Millipore) using electroblotting. The membranes were blocked in Tris-buffered saline solution containing 5% powdered milk, 1% bovine serum albumin, 0.05% Tween 20 for 1 h, incubated in the same solution with the anti-leptin antibody, washed, and then incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase. The signals corresponding to leptin were detected using the ECL system (Amersham). Conditioned medium of HEK-293 cells expressing the recombinant leptin protein (8) was used as a positive control. The Western blots were scanned by a densitometer (LKB).


RESULTS

AD-5075 Reduced Hyperglycemia and Hypertriglyceridemia in db/db Mice and ZDF Rats

Treatment of ZDF rats with AD-5075 for 4 weeks greatly reduced hyperglycemia and hypertriglyceridemia (Table 1). Likewise, AD-5075 was effective in lowering plasma glucose and triglyceride levels in db/db mice. The agent did not affect plasma glucose levels in the lean littermates of ZDF rats or db/db mice, however, it caused significant reduction of plasma triglyceride levels in both groups of lean animals. Therefore, AD-5075 exhibited anti-hyperglycemic and anti-hypertriglyceridemic efficacy in the two rodent models tested in this study.



Down-regulation of ob Gene Expression in Animals Treated with AD-5075

Northern blot analyses of RNA from white adipose tissue indicated that, compared to control lean rats, untreated obese ZDF rats expressed a 3-fold higher level of ob mRNA (Fig. 1). Following treatment with AD-5075, this level was reduced to that detected in the untreated lean animals. Moreover, when Zucker lean littermates were treated with AD-5075, a profound reduction of ob mRNA levels (3-fold) was also observed (Fig. 1). These results demonstrate that expression of the ob gene was subject to regulation by the antidiabetic agent not only in a rodent model of NIDDM, but also in the lean controls. In contrast to the down-regulation of ob mRNA expression by AD-5075, the compound significantly increased mRNA levels of adipocyte FABP in both lean and ZDF rats (Fig. 1). This is in agreement with earlier studies which demonstrated the regulatory effect of these insulin-sensitizing agents on FABP gene expression in ob/ob mice and 3T3-L1 cells(17) . We attempted to measure the level of leptin protein in plasma samples from lean and ZDF rats with or without AD-5075 treatment, but were unsuccessful due to the cross-reactivity of the antibodies used in this study with proteins in rat plasma.


Figure 1: AD-5075 down-regulates ob mRNA and up-regulates FABP mRNA levels in ZDF rats. Zucker lean rats (+/?) and ZDF rats (fa/fa) were treated with AD-5075. Total RNA was prepared from white adipose tissue and analyzed on Northern blots using cDNA probes specific for ob, adipocyte FABP, and beta-actin. A, each lane contained 10 µg of total RNA from an individual animal. B, the signals for ob and FABP were quantitated with a PhosphorImager and normalized against the signals for beta-actin (n = 5 each for lean and lean + AD, n = 6 for ZDF, and n = 8 for ZDF + AD). PIU, PhosphorImager units. *, p < 0.05 comparing AD-5075 treated animals to their respective controls.



The effect of AD-5075 on ob gene expression was further studied in db/db mice, another rodent model of NIDDM. Similar to ZDF rats, db/db mice expressed a 7 times higher level of the ob mRNA compared to the lean animals. Treatment with AD-5075 resulted in a 78% reduction of the ob mRNA level (Fig. 2). To determine the circulating level of leptin in db/db mice following chronic administration of AD-5075, plasma samples from control and treated mice were analyzed by Western blotting using affinity purified polyclonal antibodies directed against a C-terminal peptide derived from the mouse ob sequence (Fig. 3). db/db mice had 12-fold higher level of plasma leptin relative to that of the lean controls. Treatment of db/db mice with AD-5075 for 4 weeks resulted in an 86% reduction of plasma leptin concentration, with final leptin level approaching that found in the lean littermates. We attempted to measure plasma leptin protein level and ob mRNA level in white adipose tissue of lean mice following treatment with AD-5075 for 1 week but did not detect any significant changes (data not shown). Since the control lean mice expressed low amounts of ob mRNA in adipose tissue ( Fig. 2and (7) ) and had very low levels of circulating leptin as shown in Fig. 3, detection of further reduction of these parameters was hindered by the sensitivities of Western and Northern blotting. Therefore, the question of whether AD-5075 affected ob gene expression in lean animals was better addressed in the rat model. Taken together, the results indicate that the expression of the ob gene is up-regulated in obese-diabetic states and that such overexpression of the ob gene can be attenuated by treatment with an antidiabetic agent. Moreover, the agent also causes reduction of ob gene expression in the Zucker lean animals.


Figure 2: AD-5075 reduces ob mRNA level in db/db mice. AD-5075 was administered to db/db mice for 4 weeks. Total RNA was isolated from white adipose tissue of the lean controls (+/?), db/db controls, and db/db treated with AD-5075. A, the RNA samples were subjected to Northern blot analyses to determine the levels of ob mRNA. Each lane contained 10 µg of total RNA from an individual animal. B, the signals were quantitated with a PhosphorImager and normalized against the signals for beta-actin as in Fig. 1(n = 4 for each group). PIU, PhosphorImager units. *, p < 0.01 comparing db/db + AD-5075 to db/db controls.




Figure 3: AD-5075 decreases plasma leptin level in db/db mice. Plasma proteins from lean control mice (+/?), db/db control mice, and db/db mice treated with AD-5075 were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting using antibodies directed against a peptide derived from ob cDNA sequence. The signals were detected using the ECL system. A, the lane labeled as HEK-293 CM contained 10 µl of conditioned medium of 293 cells expressing a recombinant leptin(8) . The rest of the lanes each contained 6 µl of plasma from an individual animal. B, the Western blots were analyzed with a densitometer and the intensity of the signals were quantitated (n = 5 for each group). *, p < 0.02, comparing db/db versus db/db + AD.



Reduction in ob Gene Expression Is Accompanied by Changes in Body Weights

To investigate the relationships of ob gene expression with feeding behavior and weight change, food consumption and body weight were measured during the treatment with AD-5075. During the course of this study, the AD-5075-treated Zucker lean rats gained more weight and consumed more food compared to the control animals. At the end of the study, mean body weight was increased by 11.4% for the control group and 16.3% for the treated group (Table 2). The food intake was unchanged for the control lean animals but increased by 18.8% for the AD-5075-treated lean rats (Table 2). Therefore, the 70% reduction of the ob mRNA level in the lean rats as a result of treatment with AD-5075 correlated with significant increases in weight and food consumption. These data are consistent with a role of ob in the balance of caloric intake and expenditure and the control of body weight.



Decrements of the ob mRNA levels in the obese ZDF rats and the ob mRNA as well as circulating leptin levels in db/db mice were accompanied by substantial weight gains after administration of AD-5075 for 4 weeks. Mean body weight was increased by 38.8 ± 1.6% for AD-5075-treated ZDF rats (n = 8) versus 2.7 ± 1.0% for the untreated controls (n = 7), and 16.5 ± 1.8% for AD-treated db/db mice versus 7.8 ± 1.1% for untreated controls (n = 10 for each group) (Table 2). However, unlike the Zucker lean rats, there was no significant change in food consumption for db/db mice or ZDF rats treated with AD-5075 (Table 2).

When lean (+/?) mice were treated for 4 weeks with AD-5075 (5 mg/kg/day), there was also a trend toward increased weight gain. The AD-treated lean mice had a tendency to consume more food during the first 2 weeks of treatment. However, the changes were not statistically significant at the termination of the experiment. The lack of significant weight gain and hyperphagia may correlate with the lack of detectable reduction of the circulating leptin protein level and the adipose ob mRNA level in the lean mice.


DISCUSSION

The maintenance of energy balance and body weight requires a complex network involving central nervous system regulation of food intake, hormonal regulation of metabolism, energy expenditure, and ultimately energy storage in adipose tissue(18) . Parabiosis studies have suggested that the product of the ob gene may function as a circulating factor which provides negative feedback to the satiety center of the hypothalamus(19) . In addition, such studies have suggested that db/db mice and obese Zucker rats are able to produce the regulatory factor but are deficient in responding to it (20, 21) . This concept is supported by our data which demonstrate markedly increased levels of ob mRNA and leptin in these rodents. In this study, we demonstrate that treatment with an insulin-sensitizing agent of the thiazolidinedione class down-regulated the expression of ob mRNA in the Zucker lean and ZDF rats. In addition, ob mRNA level and circulating leptin level were markedly reduced in db/db mice as a result of AD-5075 treatment. In agreement with the proposed role of the ob gene product in energy balance, the reduction of ob expression correlated with increases in food intake and body weight in the Zucker lean rats which, unlike ZDF rats and db/db mice, have an intact leptin signaling loop.

The mechanism by which AD-5075 regulates ob gene expression is not well defined at present. Recent studies have indicated that thiazolidinediones are high affinity ligands for PPAR (15) and forced expression and the activation of PPAR results in the conversion of fibroblasts to adipocytes and activation of various adipose-specific genes(22) . It is possible that AD-5075 down-regulates the expression of the ob gene directly through its binding and activation of the transcription factor PPAR. Alternatively, AD-5075 may exert its effect on ob gene expression indirectly as a result of activation of PPAR which leads to changes in levels of hormones, such as insulin, and other metabolites. The animal models of NIDDM used in the present study both manifest high levels of plasma insulin, glucose, triglycerides, and free fatty acids. It is possible that any one of these factors by itself or in combination could modulate ob gene expression through direct or indirect mechanisms. Lowering these parameters by intervention with the antidiabetic agent might then result in the reduction of ob expression. In agreement with this hypothesis, recent studies have indicated that ob gene expression is subject to regulation by glucocorticoids (9) which affect plasma glucose concentration. Of note is the fact that AD-5075 only decreased the level of triglyceride but not glucose in the Zucker lean rats. The decreased ob gene expression in these lean rats suggests that there may be a correlation between the plasma lipid level and the level of ob mRNA in adipose tissue.

Since ZDF rats and db/db mice are presumed to have defects at the level of leptin receptor or downstream signaling pathways, the effect of AD-5075 in these two rodent models suggests that the compound may regulate the expression of the ob gene through a mechanism independent of the signal transduction pathways involving the leptin receptor. The fact that there are no drug-induced changes in food consumption in either of the obese animal models in the context of weight gain, while there were profound changes in both food consumption and weight gain in the intact lean rats, is potentially important. Recent studies indicate that the ob gene product regulates body weight and adiposity through effects on appetite and metabolism (5) . The lack of hyperphagia in ZDF rats and db/db mice following the treatment with AD-5075 in this study may reflect the lack of responsiveness of these animals to the effect of leptin on suppressing appetite. It is possible that the defect in these animals may be selective for components of this system involving metabolic efficiency. On the other hand, the increased weight gain seen in the two animal models may be due to the fact that AD-5075 apparently has direct effects on fat cell differentiation and fat metabolism. Thus, thiazolidinedione derivatives are efficacious adipogenic agents in 3T3-L1 and other preadipocytes(23, 24, 25) , and such adipogenesis could be the cause of the substantial weight gain observed in db/db mice and ZDF rats following treatment with AD-5075. It is possible that some aspects of altered adipocyte metabolism occur in response to the decrement of ob gene expression. An important finding of our study was the clear demonstration that the level of ob expression is not necessarily directly proportional to the mass of the tissue. The ob gene is expressed exclusively in white adipose tissue and the level of its expression is increased in several models of obesity and decreased with fasting(8) , however, administration of AD-5075 resulted in the dissociation of weight gain from increased ob expression. Furthermore, the finding of parallel induction of adipose FABP and repression of ob by AD-5075 may have important ramifications for the mechanism(s) by which this class of agent exerting antidiabetic effect.

In conclusion, we have demonstrated that the ob gene is overexpressed in two rodent models of obesity and NIDDM, and that chronic administration of a potent antidiabetic agent results in reduction of ob gene expression in ZDF rats, db/db mice, as well as in lean rats. The results provide new insights into our understanding of the metabolic defects underlying obesity and diabetes. It is important to point out that overexpression of the ob gene has recently been reported in human obese patients(26, 27, 28) . Therefore, modulation of ob gene expression using pharmaceutical agents may represent a feasible approach for treatment of obesity and NIDDM.


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 all correspondence should be addressed. R80W250, P. O. Box 2000, Rahway, NJ 07065. Tel.: 908-594-1926; Fax: 908-594-5067.

Current address: Shering-Plough Research Inst., 2015 Galloping Hill Rd., Kenilworth, NJ 07033.

**
These three authors contributed equally to this work.

§§
Supported by National Institutes of Health Grant K08HL02564.

¶¶
Supported by National Institutes of Health Grant R37DK28082.

(^1)
The abbreviations used are: NIDDM, non-insulin-dependent diabetes mellitus; AD-5075 (AD, 5-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy] enzyl]-2,4-thiazolidinedione; ob, obese gene; PPAR, peroxisome proliferator activator receptor ; FABP, fatty acid-binding protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Drs. Greg Berger, Philip Eskola, and Gerard Kieczykowski for the synthesis of AD-5075, Dr. David Bernlohr for the FABP cDNA probe, and Dr. David Moller for helpful discussions.


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