INTRODUCTION

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HYPERTENSION IS THE MOST COMMON cardiovascular disease in the United States and is a hallmark risk factor for coronary artery disease, stroke, and congestive heart failure (15). It has been estimated that one-quarter of all adults and one-half of all individuals over the age of 65 have hypertension (systolic blood pressure >140 mmHg and diastolic blood pressure >90 mmHg) in the United States and only 47% have optimal blood pressure (<120/80 mmHg) (1, 5). The incidence of hypertension is increasing, and it is more common in men than in women (1, 5, 15). Specifically, men start to show a significant rise in the incidence of hypertension in the teenage years, whereas women generally do not experience significant hypertension until after menopause (15, 31). Furthermore, it has been estimated that one-third of U.S. women 20-74 yr of age have hypertension or are administered antihypertensive medications (16a). The mechanisms for the gender-specific differences in the development of hypertension are not completely understood; however, hormonal differences (i.e., estrogen, androgens) appear to play a role.

Components of a typical Western diet, characterized by high saturated fat and refined carbohydrate, also appear to be involved in the development of hypertension. Several recent studies (9, 25, 34), including several from our own laboratory (23, 24), have suggested that dietary factors can induce endothelial dysfunction and hypertension. Specifically, it has been recently documented that long-term consumption of a Western diet induces oxidative stress and hypertension in normal female rats (24). On the basis of these data and the notion that there may be hormone-related differences in the time course for the development of hypertension, the present study was designed to test the following hypotheses: 1) arterial hypertension can be induced by diet in both male and female animals, 2) there are hormone status differences in the time course of blood pressure elevation, and 3) diet-induced hypertension can be ameliorated by either diet or estradiol (E₂) replacement.

	METHODS

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Animals and diet. All protocols were conducted in accordance with the University of California, Los Angeles, Animal Research Committee. Inbred male, female, and ovariectomized (OVX) female Fischer rats were obtained from Harlan Sprague Dawley (San Diego, CA) at 2 mo of age. This rat model has been used in previous studies in our laboratory, as the female Fischer rat normally shows little weight gain after its maturation phase (3, 4). The rats were randomly assigned to either the low-fat, complex carbohydrate (LFCC) or high-fat (primarily saturated), refined carbohydrate (sucrose) (HFS) diet, housed four per cage with a 12-h light cycle starting at 0700 at 75-76°F, and fed ad libitum with bowls placed in the cages to ensure that all animals had access to the food. The diets were prepared in powder form by Purina test diets (Richmond, IN) and contained a standard vitamin and mineral mix and all essential nutrients. The LFCC diet (Purina 5001) is low in saturated fats and contains mostly complex carbohydrates (starch), whereas the HFS diet is high in saturated and monounsaturated fats (primarily from lard plus a small amount of corn oil) and high in refined sugar (sucrose) as previously published (23). After 2 yr (female rats) and 7 mo (male rats) on the HFS diet, some of the animals were switched to the LFCC diet for a period of 2 mo (females) or 1 mo (males). In addition, the rats were weighed weekly.

E₂ replacement. Once hypertension had been established in the OVX HFS rats at 7 mo, some of the OVX rats were given E₂ replacement therapy for 1 mo by subcutaneous pellet (0.5-mg pellets, 60-day pellets, Innovative Research, Sarasota, FL). This dose is designed to achieve a serum level of E₂ in the range of 50-100 pg/ml.

Blood pressure. Blood pressure was measured by tail plethysmography (IITC) as previously described (23, 24).

Statistical analysis. Data were analyzed using a t-test or ANOVA. When significant F values were noted using an ANOVA, post hoc analyses were performed using a Newman-Keuls multiple comparison test. Differences were considered statistically significant at P < 0.05. Values reported are means ± SE with seven to eight rats per group unless otherwise indicated.

	RESULTS

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Body weight. Body weight data are shown in Table 1. For the female rats, body weight was significantly greater in the HFS group after week 20. For the male rats, body weight became significantly greater in the HFS group after week 3. For the OVX rats, body weight was greater in the OVX LFCC group compared with the normal LFCC group at randomization and greater in the OVX HFS group compared with the HFS group after week 1. In addition, body weight was significantly greater by week 6 in the OVX HFS group compared with the OVX LFCC group. After the 2-mo and 1-mo conversion to the LFCC diet, respectively, body weight was significantly reduced in both female and male rats but did not reach the value found in the LFCC rats (P < 0.05, ANOVA; Table 1). Body weight did not decrease significantly after 1 mo of E₂ replacement.

Blood pressure. Systolic blood pressure data for the female groups are depicted in Fig. 1. Systolic blood pressure was significantly elevated in the HFS compared with the LFCC rats after 1 yr (P < 0.01). Systolic blood pressure exceeded 140 mmHg in all HFS female animals by 2 yr but remained normal in the LFCC animals. Within 2 mo after conversion to the LFCC diet, blood pressure in the formerly hypertensive female HFS group returned to normal.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Systolic blood pressure of normal female rats placed on the low-fat, complex-carbohydrate (LFCC) and high-fat (primarily saturated), refined carbohydrate (sucrose) (HFS) diets at 2 mo of age and of the animals converted to the LFCC diet for 2 mo (open bar); n = 8 observations per group. *P < 0.01 vs. LFCC.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Systolic blood pressure of normal male rats placed on the LFCC and HFS diets at 2 mo of age and of the animals converted to the LFCC diet for 1 mo (open bar); n = 8 observations per group. *P < 0.01 vs. LFCC.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Systolic blood pressure of ovariectomized (OVX) female rats placed on the LFCC and HFS diets at 2 mo of age and of the animals replaced with estradiol (E2) for 1 mo (OVX+E2); n = 8 observations per group. *P < 0.01 vs. OVX-LFCC.

	DISCUSSION

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We recently documented that prolonged consumption of a diet high in fat and refined sugar by normal female rats induces marked oxidative stress, a decrease in bioavailable nitric oxide (NO), and hypertension, independent of excess energy intake (24). Others have also noted that dietary factors can affect blood pressure. Dobrian et al. (9) documented that male rats fed a Western diet for 10 wk developed hypertension. Yoshioka et al. (34) documented that chronic lard feeding increased systolic blood pressure in the absence of excessive energy intake. In addition, Shinozaki et al. (25) fed animals a high-fructose diet and reported endothelial dysfunction and elevated reactive oxygen species generation. Finally, Vogel et al. (32) reported that a single high-fat meal can induce transient endothelial dysfunction. We undertook the present study to determine whether diet could also induce hypertension in male rats and, if so, whether there is a difference in the time course and/or magnitude of the rise in blood pressure. We noted the development of diet-induced hypertension in both female and male rats. Conversion from the HFS to the LFCC diet for a period of 1 mo in males and 2 mo in females resulted in a reversal of hypertension. The Dietary Approaches to Stop Hypertension clinical trial (2) documented that a diet low in refined sugar with reduced saturated fat and high fruit and vegetable intake decreased blood pressure in both hypertensive and normotensive individuals.

The fact that the incidence of cardiovascular diseases increases after menopause suggests that estrogen plays a protective role in the cardiovascular system. Only one in nine women has clinical evidence of coronary disease in the 45- to 65-year of age range; however, this increases to one in three in those older than 65 years of age (16a). Epidemiological studies have documented that estrogen replacement is associated with a reduction in the incidence of coronary events in postmenopausal women (29). Cross-sectional data have also documented higher blood pressures after menopause (28). However, the influence of menopause on blood pressure is not without controversy. For instance, the Framingham data (13) showed no rise in blood pressure with menopause. Use of oral contraceptives pills has in some cases been associated with an increased blood pressure (28). Animal studies have also produced discordant results, possibly stemming from the differences in experimental design, species, and doses utilized (7, 16).

The present study determined the role of gonadal hormone status in the development of diet-induced hypertension. Despite the lack of estrogen in the OVX LFCC group for 7 mo, no increase in blood pressure was noted. This suggests that, in the absence of estrogen, a LFCC diet allows for maintenance of normal blood pressure. The normal blood pressure also noted in the male LFCC group supports this contention. However, estrogen deprivation in the OVX HFS animals markedly accelerated the onset of diet-induced hypertension in female rats and simulated the rise in blood pressure noted in male rats. In addition, the observation that the blood pressure differences were greatest in the HFS-fed female rats after menopause suggests a possible protective role for estrogen and loss of this protection with menopause. As noted, a similar pattern is observed in women in the United States, where hypertension is generally not seen until after menopause, and suggests that this dietary paradigm may be applicable to human hypertension. De Meersman et al. (8) demonstrated a decrease in both resting and isometric-exercise systolic and diastolic blood pressures in postmenopausal women receiving estrogen replacement therapy (ERT), which they attributed to enhanced arteriolar distensibility, since ERT had no effect on cardiac output. Evidence suggests that estrogen has a direct effect on the vascular wall, as it has been noted that it increases transcription of the endothelial NO synthase gene (14) and increases NO synthase activity (33). Data from acute infusion of E₂ to postmenopausal women (11) and chronic ERT in both OVX rats (27) and postmenopausal women (10) have shown improved endothelium-dependent relaxation. Hayashi et al. (12) found that female rabbit aortic rings do indeed produce greater basal amounts of NO compared with males and OVX females. Furthermore, estrogen deprivation in normotensive women induces oxidative stress and endothelial dysfunction (30). Interestingly, we have shown that a HFS diet can also induce oxidative stress (24) and endothelial dysfunction (22), leading to hypertension. Thus diet may compound the effects of hormones on blood pressure. The fact that both diet (this study) and estrogen deprivation alone induce hypertension and together induce more rapid hypertension supports this contention.

The role of androgens in the regulation of blood pressure is also unclear. Young OVX spontaneously hypertensive rats (SHR) do not show an increase in blood pressure; however, testosterone administration increases blood pressure in these animals. Furthermore, castration decreases blood pressure in male SHR to the level of female SHR (20). Because the increase in blood pressure in postmenopausal women takes 5-20 yr to develop (5), lack of female hormone may not be the only contributing factor for the elevated blood pressure. The shorter time course of hypertension development in the males compared with the normal females in the present study may be, in part, related to elevated androgens in the former group. Furthermore, consistent with the increase in oxidative stress and decrease in bioavailable NO noted in the present model (24), Reckelhoff (19) has proposed a scheme by which androgens increase blood pressure by stimulating renin secretion. This increases angiotensin II levels, which promotes vasoconstriction directly and indirectly by increasing the generation of superoxide, leading to enhanced NO inactivation. Along these lines, Nickenig et al. (18) demonstrated that estrogen decreases angiotensin receptor I gene expression. Furthermore, treatment of intact SHR with an androgen receptor antagonist decreases blood pressure (21). Confounding the issue is the recent finding from Nathan et al. (17) that castrated male mice fed a Western diet have an increase in atherogenic lesion formation, but testosterone supplementation reduces lesion formation, whereas aromatase inhibition reverses the protective effect, suggesting an anti-atherogenic role of testosterone due to conversion to estrogen in endothelial cells. Evidence that both diet (C. K. Roberts, R. J. Barnard, Z. Ni, and N. D. Vaziri, unpublished observations) and androgens (26) lower brain neuronal NO synthase expression and/or activity whereas estrogen increases brain NO synthase activity (33) supports the contention that diet and androgens may have an additive effect on the development of hypertension, as it is well known that NO decreases sympathetic outflow.

Overall, diet-induced hypertension is affected by hormone status. Males and OVX females display marked hypertension much earlier in their lifespan than intact females, and estrogen and androgens appear to play a role in the time course differences noted. Conversion to a LFCC diet normalized blood pressure, independent of hormone status. In addition, hormone replacement in females was also able to normalize blood pressure. Because body weight did not significantly change in the E₂-replaced OVX HFS group, estrogen apparently does not normalize diet-induced changes in blood pressure through changes in body weight. In addition, obesity does not appear to be a major contributing factor in this model, as the OVX LFCC group weighed significantly more than normal LFCC animals, without exhibiting elevated blood pressure. Thus a LFCC diet appears to mitigate the changes in blood pressure that occur with aging and different hormonal profiles and may be effective in the prevention and treatment of hypertension.