Introduction

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It is well established that there is a strong clinical association between insulin resistance, obesity and hypertension (DeFronzo, 1990; Haffner et al., 1992; Wajchenberg et al., 1994; Ferrannini et al., 1990). However, despite this epidemiological evidence and the recognition that patients with essential hypertension are insulin resistant (Ferrannini et al., 1990; DeFronzo, 1990) the underlying pathogenic link between hypertension, obesity and insulin resistance remains uncertain. Nevertheless, there is clear evidence to suggest that hyperinsulinemia is predictive of and causal in the development of noninsulin-dependent diabetes and hypertension (DeFronzo, 1990; Wajchenberg et al., 1994). Furthermore there are several mechanisms that could explain this association, including insulin-stimulated sodium reabsorption in the kidney, stimulation of the sympathetic nervous system, alteration of membrane ion transport, altered vascular reactivity or impaired insulin dependent arteriolar vasodilatation (DeFronzo, 1990; Daly and Landsberg, 1991; Corry and Tuck, 1996).

Obesity, hyperinsulinemia and hyperlipidemia also characterize the genetic defect in the obese Zucker rat (Bray and York, 1979) and in this regard it manifests all the metabolic abnormalities that characterize the insulin resistance Syndrome X in man (Reaven, 1988; Wajchenberg et al., 1994). In addition to these perturbations in metabolism, arterial pressure has been shown to be higher in obese rats compared to their lean littermates after 6 mo of age (Kurtz et al., 1989; Kasiske et al., 1992; Turner et al., 1995). The mechanism for the elevation of blood pressure, however, is unclear, particularly because sympathetic drive (Levin et al., 1983) and plasma renin activity (Harker et al., 1993) are reported to be reduced in obese Zucker rats, but it has been proposed to be secondary to renal injury or increased renal sodium reabsorption (Kasiske et al., 1992).

Recent evidence suggests that the primary cause of obesity in the falfa Zucker rat is a mutation in the leptin receptor (Philips et al., 1996). Nevertheless, high corticosterone levels are thought to play an important role in the development and maintenance of the obesity syndrome in the Zucker rat. Thus expression of the obese phenotype can be prevented in weanling or partially reversed in postweanling obese Zucker rats by adrenalectomy (Fletcher, 1986; Freedman et al., 1986; Castonguay, 1991) and restored in adrenalectomized obese Zucker rats by corticosterone replacement (Fletcher, 1986; Freedman et al., 1986; Castonguay, 1991). Similarly, mifepristone, a type II glucocorticoid receptor antagonist (Brogden et al., 1993), has been reported to reduce obesity and hyperphagia and to ameliorate the hyperinsulinemia of young obese rats mimicking the effect of adrenalectomy (Langley and York, 1990).

It is well established that both glucocorticoids and mineralocorticoids increase blood pressure and that clinical conditions such as Cushing's syndrome characterized by raised glucocorticoid levels are associated with hypertension (Walker and Edwards, 1994). In addition, administration of corticosterone or selective type II glucocorticoid agonists to normal rats induces a reversible hypertension that is prevented or reversed by inhibition of type II, but not type I, glucocorticoid receptors (Grunfeld et al., 1985). Furthermore, sodium-independent hypertension in humans with glucocorticoid excess (Cushing's syndrome) can be controlled by mifepristone but not the type I antagonist spironolactone (Mantero and Boscaro, 1992).

There is a general lack of information regarding the pathogenesis of hypertension associated with obesity but it has been suggested that the Zucker rat might be a suitable animal model of obesity and hypertension (Kurtz et al., 1989). In view of the importance of glucocorticoids to the development and maintenance of the obese phenotype in Zucker rats and their connection with essential hypertension in man the aim of our study was to investigate the involvement of glucocorticoids in hypertension in aged obese Zucker rats. We have used the glucocorticoid II receptor antagonist mifepristone to explore the contribution of endogenous corticosterone to the maintenance of hypertension in obese Zucker rats and compared this to the effects of corticosterone-21-acetate on blood pressure in lean animals.

	Methods and Materials

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Adult male obese Zucker rats and their lean littermates were obtained from Harlan Olac (Bicester, Oxfordshire, U.K.) and fed RM1 diet (SDS, Witham, U.K.) ad libitum during the run-up to dosing and allowed free access to water at all times. All experiments were approved by the Procedures Review Panel of SmithKline Beecham Pharmaceuticals U.K. and complied with the Guidance on the Operation of the Animals (Scientific Procedures) Act 1986.

Blood pressure measurement. In view of excessive lipid deposition in indwelling cannula and poor surgical wound healing in obese Zucker rats (personal observations) blood pressure was measured noninvasively using a tail cuff plethysmographic method (Apollo model 179; IITC Life Science, Woodland Hills, CA) that has been previously validated (Bunag and Butterfield, 1984; Bunag, 1984). The animals were acclimatized to an ambient temperature of 29 to 31°C for 15 min before blood pressure determination and values were taken from the mean of at least three recordings per animal.

Study design. Rats were placed in perspex restraining tubes and housed in an incubator maintained at 29 to 32°C. After a 15-min equilibration period, blood pressure was measured using an inflatable cuff and pulse sensor, placed around the tail, coupled to a model 679 semiautomatic blood pressure system (ITTC Inc., Woodland Hills, CA). The inflated cuff pressure was 275 mmHg and pressure was released at a rate of 500 mmHg min¹. Systolic blood pressure was calculated as the mean of at least three readings. Blood pressure was monitored monthly, from 5 mo of age, until SBP in obese rats was significantly greater than their lean littermates for 2 consecutive mo. Drug treatment was initiated when the animals were 8 mo of age. Animals were randomly assigned to a control group or to a treatment group. All animals received drug vehicle (propylene glycol, 2.0 ml · kg¹ day¹) by gavage for 14 days starting at day 1. Blood pressure was measured on three further occasions on days 3, 8 and 10 to establish a baseline level of SBP. One obese and one lean group then received mifepristone (a gift from Rousell-UCLAF, Paris, France; 40.0 mg · kg¹ day¹) a second lean group received corticosterone-21-acetate (3.0 mg · kg¹ day¹); control animals (lean and obese) remained on vehicle and dosing continued once daily for another 9 days. Blood pressure measurements were made 2, 4 and 9 days after commencement of dosing.

Analysis. Whole blood was centrifuged at 3000 rpm for 10 min at 4°C. Plasma was aspirated and aliquots stored at 20°C before measurement of plasma hormones. Commercially available radioimmunoassay kits were used to measure plasma insulin (Amersham International plc, Amersham, U.K.), aldosterone (DPC Diagnostics, Caernarfon, U.K.), corticosterone (Amersham International plc) and plasma renin activity (New England Nuclear, Stevenage, U.K.). Glucose levels were determined by the glucose oxidase method from hemolyzed whole blood.

Statistical analysis. Results are expressed mean ± S.E. Statistical analysis between groups was performed by one-way ANOVA. Where significant between-group variation was observed, Dunnett's test for multiple comparisons (Wallenstein et al., 1980) was performed to identify the source of variance. All statistical analyses were performed using SAS Research Scientist Application program, version 1.4 (SAS Institute Inc., Carey, NC). P < .05 (95% level) were considered significant.

	Results

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Significant differences in systolic blood pressure between obese Zucker rats and their lean littermates were observed from 6 mo of age (table 1). Obese animals were also significantly heavier and were hyperphagic compared to their lean littermates at the start of the study at 8 mo of age (table 1). At the end of the study period, obese Zucker rats receiving vehicle gained 13 g body weight compared to a 25 g loss of weight in the mifepristone treated obese animals (table 2). Neither mifepristone nor corticosterone-21-acetate treatment significantly affected body weight compared to vehicle treated leans in this study (table 2).

View this table: [in this window] [in a new window]   TABLE 2 Plasma hormone levels after administration of mifepristone (40.0 mg · kg1 day1) for 9 days to obese and lean Zucker rats and administration of corticosterone-21-acetate (3.0 mg · kg1 day1) for 9 days to lean Zucker rats

Effects of mifepristone in lean and obese Zucker rat. Before dosing, SBP in obese rats was significantly higher than in their lean littermates (table 1). During administration of mifepristone (40.0 mg · kg¹ day¹ p.o.), SBP gradually decreased to a level of 134 ± 4 mmHg after 9 days of oral dosing although SBP in vehicle-treated obese Zucker rats was unchanged from baseline at 153 ± 4 mmHg and remained significantly higher than vehicle treated leans (132 ± 5 mmHg; fig. 1). In lean rats, SBP was not significantly affected by mifepristone (fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Systolic blood pressure measurement before and during treatment of obese Zucker rats with propylene glycol (2.0 ml · kg1 day1 p.o., n = 7-9 ), mifepristone (40 mg · kg1 day1 p.o., n = 9 ) and lean Zucker rats treated with propylene glycol (2.0 ml · kg1 day1 p.o., n = 8 ). Drug treatment phase indicated by solid box. Values are mean ± S.E. *P < .05 vs. obese vehicle.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Systolic blood pressure measurement before and during treatment of lean Zucker rats with propylene glycol (2.0 ml · kg1 day1 p.o., n = 8 ), mifepristone (40 mg · kg1 day1 p.o., n = 9 ) and obese rats treated with propylene glycol (2.0 ml · kg1 day1 p.o., n = 7-9 ). Drug treatment phase indicated by solid box. Values are mean ± S.E. *P < .05 vs. lean vehicle.

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Effect of corticosterone 21 acetate in lean Zucker rats. Administration of corticosterone-21-acetate (3.0 mg · kg¹ day¹) to lean Zucker rats induced a gradual rise in SBP from 133 ± 3 mmHg at the start of dosing to 150 ± 15 mmHg after 9 days of once daily oral dosing (fig. 3). SBP in vehicle-treated leans remained significantly lower than their obese littermates tending to decline slightly during the course of the experiment (fig. 3).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Systolic blood pressure measurement before and during treatment of lean Zucker rats with propylene glycol (2.0 ml · kg1 day1 p.o., n = 8 ), corticosterone-21-acetate (3.0 mg · kg1 day1 p.o., n = 6 ) and obese rats treated with propylene glycol (2.0 ml · kg1 day1 p.o., n = 7-9 ). Drug treatment phase indicated by solid box. Values are mean ± S.E. *P < .05 vs. lean vehicle.

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	Discussion

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Numerous studies have shown that glucocorticoids are crucial to the development of obesity in animal models (Bray and York, 1979; Bray et al., 1990) and for the full expression of the metabolic disorders which characterize the obese Zucker rat (Freedman et al., 1986; Langley and York, 1990; Castonguay, 1991). In man, abdominal obesity and increased viceral fat mass are associated with hypertension and it has been suggested that the link between the two is insulin resistance and hyperinsulinemia (Kissebah and Krakower, 1994). Previous studies in obese Zucker rats have shown that both adrenalectomy and the antiglucocorticoid mifepristone inhibits the development of obesity and ameliorates the attendant hyperinsulinemia and hyperlipidemia (Freedman et al., 1986; Langley and York, 1990; Alarrayed et al., 1992). In our study we have demonstrated that from 6 mo of age obese rats are hypertensive compared to their lean littermates and that this increase in systolic blood pressure is reversed by treatment with mifepristone. Mifepristone is a type II glucocorticoid receptor antagonist that does not bind to the type I mineralocorticoid receptor or to aldosterone receptors, but that also is an antagonist at progesterone and androgen receptors (Brogden et al., 1993). The similarity between the effects of mifepristone and adrenalectomy on obesity in the obese Zucker rat and in dietary models, suggests that it is the type II receptor rather than progesterone receptors that regulates the development of obesity (Langley and York, 1990; Okada et al., 1992). Similarly in our experiments our observation that the blood pressure of lean Zuckers could be elevated to levels seen in obese animals by treatment with corticosterone-21-acetate together with its reduction in obese animals by mifepristone, lends support to the hypothesis that it is also the type II receptor that is involved in hypertension in the obese animal.

The mechanisms mediating hypertension in the obese Zucker is unclear. Previous studies (Levin et al., 1983; Kasiske et al., 1992) indicate that the obese Zucker has diminished sympathetic nervous system activity and plasma renin activity is not raised (this study) or is reduced (Harker et al., 1993). Nevertheless, both angiotensin-converting enzyme inhibitors and angiotensin II antagonists reduce blood pressure in obese Zucker rats (Schmitz et al., 1992; Crary et al., 1995). In this regard it is interesting to note that mifepristone has been reported to inhibit both glucocorticoid-induced hypertension in rats and angiotensinogen synthesis (Agarwal et al., 1987).

In NIDDM subjects there is a positive correlation between insulin levels and blood pressure (Corry and Tuck, 1996) and there are a number of mechanisms by which hyperinsulinemia might mediate hypertension (Corry and Tuck, 1996). Furthermore agents that improve insulin responsiveness such as the insulin-sensitizing thiazolidinediones, CS 045 and ciglitazone, are reported to be antihypertensive in the obese Zucker rat (Yoshioka et al., 1993; Pershadsingh et al., 1993). The effects of mifepristone on blood pressure in our study, however, appeared independent of hyperinsulinemia as judged by the unchanged fasting plasma insulin levels.

A surprising finding of our study was that plasma aldosterone levels were greater in obese animals than their lean counterparts and were reduced to lean levels by mifepristone. The mechanism by which mifepristone reduced, and corticosterone-21-acetate increased, plasma aldosterone levels, in the absence of changes in PRA is uncertain but suggests a mechanism independent of renal renin secretion. In support of this, weight gain in the dog has been reported to be associated with an increase in plasma aldosterone without changes in plasma renin activity (Rocchini et al., 1989) and increased plasma aldosterone concentrations despite normal plasma renin activity has been reported in obese man (Ljutic et al., 1995). Adrenal renin and angiotensin II levels are reported to be high (Inigami et al., 1989; Philips et al., 1993) and it is known that there is an intrinsic renin-angiotensin system within the adrenal cortex that regulates aldosterone secretion (Gupta et al., 1995, Mulrow and Franco-Saenz, 1996). It is hypothesized therefore that the adrenal renin-angiotensin system provides a local mechanism for the regulation of aldosterone secretion (Mulrow and Franco-Saenz, 1996). Indeed it is known that ACTH stimulation of aldosterone production is reduced by ACE inhibitors (Ramirez et al., 1988). Our current data suggest that the renin-angiotensin system in the adrenals may also be regulated by glucocorticoids. It is possible that glucocorticoids up-regulate expression of the angiotensin AT₁ receptor as is reported in the vasculature (Provencher et al., 1995) increasing the responsiveness of the aldosterone secreting cells to locally produced angiotensin II and to ACTH. In addition an interaction between glucocorticoids and the adrenal renin-angiotensin system may provide a possible mechanism to explain the antihypertensive effects of both the antiglucocorticoid mifepristone and ACE inhibitors or angiotensin II antagonists in obese Zuckers. This hypothesis places aldosterone as an important contributor to elevated blood pressure in the obese Zucker and in this regard it is interesting to note that resting plasma aldosterone concentrations are also reported to be one of the contributing factors responsible for the elevation of blood pressure in obese subjects (Ljutic et al., 1995). Nevertheless hypertension in the obese animal is likely to be a multifactorial process and other factors such as inhibition of the effects of glucocorticoids on vascular tone and responsiveness may be involved in the antihypertensive effects of mifepristone in these experiments.

Our data and that of others support a connection between raised circulating glucocorticoid levels, obesity and hypertension in Zucker rats. Although there is little question about the association between abdominal obesity and increased blood pressure and insulin resistance in man (Kissebah and Krakower, 1994) their association with elevated cortisol secretion is less certain. However, there is increasing evidence that abdominal fat deposition is linked to hyperreactivity of the hypothalamic-pituitary-adrenal axis (Marin et al., 1992; Pasquali et al., 1993) and both cortisol and aldosterone secretion are reported to be elevated in obese subjects (Marin et al., 1992; Ljutic et al., 1995). Although there is strong evidence to connect increased hypothalamic-pituitary-adrenal axis activity with obesity and insulin resistance (Kissebah and Krakower, 1994), our data are the first to show that the associated hypertension may also be a consequence of the dysregulation of adrenal steroid secretion.

We conclude that corticosterone contributes to the maintenance of hypertension in the obese Zucker rat through mechanisms that might be secondary to increased aldosterone secretion. Our data suggest that raised glucocorticoids and aldosterone may be factors contributing to hypertension in obesity.