(Received for publication, August 1, 1995; and in revised form, January 30, 1996)
From the
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.
Obesity is a predisposing factor for insulin resistance and
non-insulin-dependent diabetes mellitus (NIDDM) ()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.
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
[-
P]deoxy-CTP.
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 -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
-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 -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.
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.
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.