Introduction

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The prevalence of hypertension in humans increases with obesity (Hall, 1994). In the central nervous system, the hypothalamus is an important structure that regulates food intake and blood pressure. Thus, hypothalamic lesions induce not only obesity, but also sex-related differences in blood pressure control (Baylis et al., 1996). Norepinephrine in the hypothalamus plays a potential role in the regulation of blood pressure (Philippu et al., 1973). An increase in hypothalamic norepinephrine release has been observed in spontaneously hypertensive rats (Meldrum and Westfall, 1986), and the hypothalamic activity of the monoamine biosynthetic enzyme, tyrosine hydroxylase (TH), was found to be increased in this genetic model of hypertension (Nagaoka and Lovenberg, 1977). In synapses, norepinephrine release is inhibited following activation of presynaptic ₂-adrenoceptors (Starke, 1987). In both human and rat, pharmacological and molecular studies of ₂-adrenoceptors have suggested that at least three distinct genes that encode three distinct subtypes, designated _2A, _2B, and _2C (Bylund, 1992), are all expressed in the brain (Zeng and Lynch, 1991; Tavares et al., 1996). Using genetically engineered mice with disrupted genes, it has been shown that the _2A-adrenoceptor subtype plays a crucial role in the central hypotensive response to ₂-adrenoceptor agonists (MacMillan et al., 1996) and that the inhibitory presynaptic ₂-adrenoceptor belongs predominantly to this subtype (Altman et al., 1999; Starke, 2001). In contrast, the _2B-adrenoceptor mediates the ₂-agonist-induced increase in blood pressure (Link et al., 1996). Moreover, from a subsequent series of studies on salt-induced hypertension in ₂-adrenoceptor subtype-deficient mice (Makaritsis et al., 1999a,b), or by using the antisense strategy (Kintsurashvili et al., 2001), it has been suggested that the adrenergically mediated hypertension depends on the central _2B-adrenoceptor subtype. In contrast with other brain structures, hypothalamus is the area in which the _2B-adrenoceptor gene is the subtype most expressed (Tavares et al., 1996). Altogether, these observations tend to attribute an important role to hypothalamic _2B-adrenoceptors in the onset of hypertension.

Cafeteria diet feeding leads to a gender-related difference in obesity-induced hypertension (Coatmellec-Taglioni et al., 2002). In fact, only male cafeteria-fed rats developed hypertension. As in obese subjects, the prevalence of cardiovascular disease is greater in men than women, female sex hormones are generally considered as protective in women before menopause (Lönnqvist et al., 1997). Maturation of the noradrenergic system in brain is relevant to the effects of gonadal steroids at birth, and neonatal androgenization of female rats induces a male-type development of this system in the hypothalamus (Simerly, 1989; Siddiqui and Shah, 1997).

The aim of the present study was to determine whether cafeteria diet feeding alters differently the hypothalamic noradrenergic system and, especially, the expression of genes encoding the TH and the different ₂-adrenoceptor subtypes in male and female rats. To establish a possible link between neonatal androgenization and susceptibility to cafeteria-diet induced hypertension, this study was further extended to the neonatal testosterone-imprinted female.

	Experimental Procedures

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Animals, Diet, and Study Procedure. Nulliparous time-mated Sprague-Dawley female rats (day 20 of pregnancy) were purchased from Centre d'Elevage de Rats Janvier (Le Genest St Isle, France), maintained at constant room temperature (24°C) on a 12-h light/dark cycle, and housed in individual cages until parturition. At birth, female pups were divided into two groups receiving (within 3 h after birth) one subcutaneous injection of either 1 mg of testosterone propionate in olive oil (50 µl, n = 8) or vehicle alone (n = 10) according to the method of Nilsson et al. (1998). Male (n = 12), female, and testosterone-treated female pups were raised in equal groups with lactating mothers until 22 days of age. At this age, rats were divided into two subgroups, housed in individual cages, and allowed free access to either standard laboratory chow (25% protein, 6% fat, and 69% carbohydrate; UAR, Epinay-sur-Orge, France) or cafeteria diet (15% protein, 49% fat, and 36% carbohydrate) as previously reported (Coatmellec-Taglioni et al., 2000). After 10 weeks of diet, systolic blood pressure was measured between 10:00 AM and 12:00 PM by using the tail-cuff method with an electrosphygmomanometer (Physiograph MK-II; Narco-Bio-Systems Inc., Houston, TX), on unanesthetized, restrained rats warmed to 38°C for 10 min. At least five replicate blood pressure measurements were checked on two consecutive days (10 measurements over 2 days). For each individual, the average of all measurements was taken as representative of its systolic blood pressure. Two days later, and after an overnight fast, rats were killed by decapitation, and their trunk blood was immediately collected in heparinized tubes. After centrifugation, plasma was separated and stored at 80°C for hormonal determinations. Hypothalami were carefully dissected, rapidly frozen in liquid nitrogen, and stored at 80°C. All experimental protocols were approved by the University Animal Use and Care Committee.

Hormonal Determinations. Plasma leptin levels were measured by radioimmunoassay using a commercial kit provided by Linco Research Inc. (St. Charles, MO) with rat leptin as standard and according to the protocol of the manufacturer.

RNA Isolation and cDNA Synthesis. Total hypothalamic RNA was isolated using guanidium thiocyanate-phenol-chloroform extraction, with the TRIzol reagent (Invitrogen, Cergy-Pontoise, France). Rat hypothalamic RNA (3 µg) was treated with 3 units of DNase I (Invitrogen) for 25 min at 25°C. After denaturation of DNase I at 95°C for 5 min, RNA-directed cDNA synthesis was performed with Superscript II reverse transcriptase and oligo(dT)_12-18 primer (Invitrogen) for 50 min at 42°C according to the manufacturer's protocol. Then, mixture was incubated for 10 min at 70°C for reverse transcriptase denaturation and diluted in 4 volumes of sterile water, and the synthesized cDNA was used for PCR experiments.

PCR Experiments. Each PCR (100 µl final volume) was carried out with 10 µl of cDNA as template, in the presence of HotStarTaq DNA polymerase (QIAGEN S. A., Courtaboeuf, France), under the conditions recommended by the supplier, and 1 µCi of [³H]dCTP. PCR mixtures of cDNA and the respective primers (Table 1) were amplified using a program temperature control system (Appligen Oncor, Illkirch, France). One PCR cycle consisted of 1 min at 94°C, 1 min at 57°C, and 1 min at 72°C for ₂-adrenoceptor cDNA subtypes with numbers of amplification cycles of 33 for the _2B- and 36 for _2A- and _2C-subtypes. For -actin, similar experimental conditions were used, except that the annealing temperature was 53°C and the number of amplification cycles was 26. Primers used for -actin amplification were chosen to span two introns to discriminate the cDNA amplification products from genomic DNA contamination. For TH cDNA amplification, we used an annealing temperature of 56°C and an elongation time of 1 min 30 s, and the number of amplification cycles was 30. Each reaction mixture was resolved in a 1.5% low melting point agarose gel (Invitrogen) stained with ethidium bromide and documented on Polaroid 665 film (Polaroid UK Ltd., St. Albans, UK). For quantification, respective bands of interest were excised and melted at 70°C, and the incorporated radioactivity was determined by scintillation counting in Ready Safe (Beckman Instruments France S.A., Gagny, France).

Materials. [³H]dCTP (1.92 × 10¹² Bq/mmol) and DNA molecular weight markers (100-bp ladder) were purchased from Amersham Biosciences (Les Ullis, France). Primers were synthesized by Eurogentec (Horstal, Belgium). All other materials were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

Result Expression and Statistical Analysis. All results were expressed as the means ± S.E.M. Statistical analysis of physiological parameters was performed by analysis of variance followed by the Student-Newman-Keuls multiple comparison test. For DNA amplification, the incorporated radioactivity was normalized with respect to the length of each cDNA, and mRNA levels were expressed versus -actin mRNA content. Results are expressed as percentages of their respective control group, and statistical analyses were performed by the Student t test. P < 0.05 was considered statistically significant.

	Results

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Effects of Cafeteria Diet Feeding on Physiological Parameters. Male and intact female rats fed the standard chow were normotensive, and neonatal testosterone treatment did not change blood pressure in female rats (Table 2). Cafeteria diet feeding for 10 weeks induced a sexual dimorphism in systolic blood pressure, which was found to be either significantly increased by the diet in males or normal in intact females (Table 2). However, this gender difference disappeared when females were treated at birth with testosterone. Indeed, in the cafeteria diet-fed groups, testosterone-treated female rats had higher systolic blood pressure increases than males (Table 2). As shown in Table 2, no significant differences in body weight were observed between intact and neonatal testosterone-treated females fed the standard diet. The cafeteria diet increased body weights, fat mass/body mass ratio, and plasma leptin levels whatever the sex and treatment. No significant differences in fat mass/body mass ratio and leptin levels were found among the three groups of standard diet-fed rats or among the three groups of cafeteria diet-fed rats (Table 2).

Hypothalamic Tyrosine Hydroxylase and ₂-Adrenoceptor Gene Subtype Expressions. Experiments using reverse transcription-polymerase chain reaction (RT-PCR) were performed with specific TH and ₂-adrenoceptor subtype primers to determine whether cafeteria diet is associated with modifications in mRNA levels. cDNA amplifications with these specific primers provided amplified products of the predicted sizes [1123 bp for TH (Fig. 1, top), 311 bp for _2A-adrenoceptor (Fig. 2, top), 407 bp for _2B-adrenoceptor (Fig. 3, top), and 426 bp for _2C-adrenoceptor (Fig. 4, top)] in all groups of rats. It must be noted that the fragment generated from -actin primers (280 bp; Fig. 1, middle), which is present at comparable levels between standard and cafeteria diet in each group of rats, is the only fragment amplified, thus ruling out any genomic DNA contamination in these experiments. After 10 weeks of cafeteria diet feeding, analysis of the radioactivity incorporated in the hypothalamic products normalized to -actin revealed a dramatic increase in TH mRNA levels (+422%; Fig. 1, bottom) in males compared with their respective controls fed the standard diet. Although weaker (+95%), significant increase in TH mRNA levels was also observed after cafeteria diet feeding in neonatal testosterone-treated females compared with their respective controls fed the standard chow. In contrast, no changes in hypothalamic TH mRNA levels were found in intact females regardless of the diet. Compared with standard chow, cafeteria diet increased _2A-adrenoceptor mRNA levels in intact female rats (Fig. 2, bottom), but not in male and testosterone-treated female rats. In contrast, cafeteria diet feeding up-regulated only _2B-adrenoceptor mRNA levels in both male and testosterone-treated female rats (Fig. 3, bottom). Moreover, cafeteria diet induced an increase in _2C-adrenoceptor mRNA levels in male rats but no significant changes in both groups of females (Fig. 4, bottom).

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Hypothalamic gene expression of tyrosine hydroxylase and -actin. A, ethidium bromide staining of RT-PCR products using TH or -actin primers in hypothalamus of male, female, and testosterone-treated female rats fed standard and cafeteria diet. Results are representative of one experiment performed as described under Experimental Procedures. Lanes 1 and 8, DNA size markers; lane 2 (male), lane 4 (female), and lane 6 (testosterone-treated female), rats fed standard diet; lane 3 (male), lane 5 (female), and lane 7 (testosterone-treated female), cafeteria-fed rats. Upper panel, TH products; lower panel, -actin products. B, gene expression of tyrosine hydroxylase normalized to -actin levels and expressed as a percentage of their respective controls. Data are given as the mean ± S.E.M. for males (n = 6), females (n = 5), and testosterone-treated females (n = 4). , P < 0.01; and , P < 0.001 for the comparison between controls and cafeteria-fed rats.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Hypothalamic gene expression of 2A-adrenoceptor. A, ethidium bromide staining of RT-PCR products using 2A-adrenoceptor primers in hypothalamus of male, female, and testosterone-treated female rats fed standard and cafeteria diets. Results are representative of one experiment performed as described under Experimental Procedures. Lanes 1 and 8, DNA size markers; lane 2 (male), lane 4 (female), and lane 6 (testosterone-treated female), rats fed standard diet; lane 3 (male), lane 5 (female), and lane 7 (testosterone-treated female), cafeteria-fed rats. B, gene expression of 2A-adrenoceptor normalized to -actin levels and expressed as a percentage of their respective controls. Data are given as the mean ± S.E.M. for males (n = 6), females (n = 5), and testosterone-treated females (n = 4). , P < 0.05 for the comparison between controls and cafeteria-fed rats.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Hypothalamic gene expression of 2B-adrenoceptor. A, ethidium bromide staining of RT-PCR products using 2B-adrenoceptor primers in hypothalamus of male, female and testosterone-treated female rats fed standard and cafeteria diets. Results are representative of one experiment performed as described under Experimental Procedures. Lanes 1 and 8, DNA size markers; lane 2 (male), lane 4 (female), and lane 6 (testosterone-treated female) rats fed standard diet; lane 3 (male), lane 5 (female), and lane 7 (testosterone-treated female) cafeteria-fed rats. B, gene expression of 2B-adrenoceptor normalized to -actin levels and expressed as a percentage of their respective controls. Data are given as the mean ± S.E.M. for males (n = 6), females (n = 5), and testosterone-treated females (n = 4). , P < 0.05 for the comparison between controls and cafeteria-fed rats.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Hypothalamic gene expression of 2C-adrenoceptor. A, ethidium bromide staining of RT-PCR products using 2C-adrenoceptor primers in hypothalamus of male, female, and testosterone-treated female rats fed standard and cafeteria diets. Results are representative of one experiment performed as described under Experimental Procedures. Lanes 1 and 8, DNA size markers; lane 2 (male), lane 4 (female), and lane 6 (testosterone-treated female) rats fed standard diet; lane 3 (male), lane 5 (female), and lane 7 (testosterone-treated female), cafeteria-fed rats. B, gene expression of 2B-adrenoceptor normalized to -actin levels and expressed as a percentage of their respective controls. Data are given as the mean ± S.E.M. for males (n = 6), females (n = 5), and testosterone-treated females (n = 4). , P < 0.01 for the comparison between controls and cafeteria-fed rats.

	Discussion

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Our present results show that neonatal testosterone-imprinted female and male rats exhibit a significant increase in their systolic blood pressure after 10 weeks of cafeteria diet feeding compared with their respective controls fed a standard chow. This phenomenon does not appear in intact female rats. In spontaneously hypertensive rats, a previous study reported that the hormone imprinting occurring at birth contributes to the sexually dimorphic pattern of hypertension (Cambotti et al., 1984). From the present investigation, it appears clearly that neonatal treatment of female rats with testosterone, just as naturally occurring testosterone in males (Bain, 1983), has induced susceptibility to develop hypertension when these animals are fed the cafeteria diet.

Based on RT-PCR studies, the cafeteria diet increased hypothalamic mRNA levels of the rate-limiting enzyme of catecholamine biosynthesis, TH, only in the hypertensive male and testosterone-treated female rats. TH activity is regulated by short-term mechanisms such as the phosphorylation of the enzyme and long-term processes including modulation of gene transcription and protein synthesis (Stachowiak et al., 1990). One of the consequences of such an increased TH activity, as observed in both male and testosterone-treated female rats, is a higher norepinephrine secretion that could contribute to the development of a hyperadrenergic state and, thus, to the maintenance of elevated blood pressure. Interestingly, it has been shown that leptin up-regulates TH gene expression in adrenals (Takekoshi et al., 1999) and that a single leptin injection in the ventromedial hypothalamus increases plasma norepinephrine in a dose-dependent manner (Satoh et al., 1999). In the present study, all groups of cafeteria-fed rats developed hyperleptinemia. However, TH gene expression was unchanged by the cafeteria diet in intact female rats. Indubitably, the hypothalamic relationship between leptin, female gonadal steroids, and TH gene expression remains to be determined in the cafeteria-fed rat, a model of obesity, and more generally in obesity, since this pathology is assumed to represent a state of central leptin resistance (Scarpace et al., 2001).

The other major finding of the present study was that cafeteria diet induced differential increases in the hypothalamic ₂-adrenoceptor mRNA subtype levels, depending on the sex and the neonatal testosterone treatment of the animals. Indeed, whereas an _2B-adrenoceptor over-expression occurred in the hypertensive male and testosterone-treated female rats, the _2A-adrenoceptor gene expression was, in contrast, increased only in the normotensive cafeteria-fed females. Intriguingly, cafeteria diet increased _2C-adrenoceptor mRNA levels solely in the hypertensive male rats. A potential role for the _2B-adrenoceptor in the development of high blood pressure is strengthened by the following studies. In _2B-adrenoceptor knockout mice, a lack of immediate hypertensive response to ₂-adrenoceptor agonists has been reported (Link et al., 1996). Moreover, various experiments on salt-induced hypertension in mice deficient in one of the three ₂-adrenoceptor subtypes (Makaritsis et al., 1999a,b, 2000) allowed the suggestion that adrenergically mediated hypertension depends on the central _2B-adrenoceptors. The validity of this hypothesis is further supported by a recent study showing that intracerebroventricular microinjection of antisense-oligonucleotides targeting a specific sequence of the _2B-adrenoceptor gene in salt-sensitive rats lowered blood pressure (Kintsurashvili et al., 2001). Therefore, it seems likely that the up-regulation of hypothalamic _2B-adrenoceptor gene expression that we observed only in the hypertensive cafeteria diet-fed rats participates in the maintenance of the hypertensive state. It is noteworthy that we have recently reported similar gender differences in renal _2B-adrenoceptor gene expression in the cafeteria-fed rat, a model of obesity-related hypertension (Coatmellec-Taglioni et al., 2000, 2002). Thus, from the latter and present studies, it appears that both peripheral and central over-expression of the _2B-adrenoceptor gene could be a determinant factor contributing to the hypertensive pattern of the cafeteria diet-fed rats. On the other hand, the up-regulation of the _2A-adrenoceptor gene expression in normotensive cafeteria-fed female rats could reflect an adaptive and protective reaction of these animals in response to the cafeteria diet. Supporting this view is a recent report on knockout mice providing clear evidence that the _2A-adrenoceptor is the subtype mediating the hypotensive effect of ₂-adrenoceptor agonists (MacMillan et al., 1996; Altman et al., 1999). Moreover, these _2A-adrenoceptor knockout mice have altered sympathetic regulation due to the loss of _2A-adrenoceptor-mediated inhibition of catecholamine release from sympathetic nerve terminals (Altman et al., 1999). Thus, the up-regulation of the _2A-adrenoceptor gene expression observed only in the intact females could reinforce the control of central sympathetic outflow during the cafeteria diet feeding and, besides, could explain why these animals have a normal blood pressure. Conversely, hypertension of cafeteria-fed rats could be the consequence of the absence in males, or of the loss in androgenized females, of this adaptive change. One intriguing observation is the increase in _2C-adrenoceptor mRNA levels that we found only in male cafeteria-fed rats. So far, all studies on knockout mice are concordant to indicate that the _2C-adrenoceptor is not involved in blood pressure regulation (Link et al., 1996; MacDonald et al., 1997). It has been shown that the inhibitory effect on norepinephrine release of ₂-autoreceptors is predominantly mediated by presynaptic _2A-adrenoceptors and, for a minor part, by the _2C-subtype (Starke, 2001). It is thus conceivable that the increase in _2C-adrenoceptor gene expression seen only in males reflects a compensatory response to higher sympathetic activity. This effect does not prevent the onset of hypertension but could limit the increase in blood pressure, as shown by the higher hypertension observed in testosterone-treated female than in male rats. Hypothalamic areas are important in the regulation of blood pressure. In fact, the anterior hypothalamus is generally considered as a depressor area (Gellman et al., 1981). This is illustrated by the finding that microinjection of the selective ₂-agonists clonidine and guanabenz into the anterior hypothalamus produces depressor and bradycardic responses that are both antagonized by rauwolscine (Wyss et al., 1988). In the opposite way, electrical stimulation of the posterior hypothalamus induces pressor responses with increasing sympathetic nerve activity (Takeda and Bunag, 1978). Norepinephrine contributes to eliciting the pressor responses following electrical stimulation (Philippu et al., 1973). Our present results obtained in whole hypothalamus call for future studies on distribution and function of ₂-adrenoceptor subtypes in each hypothalamic area to clarify their respective contribution to cafeteria diet-induced hypertension.

As shown herein, the resistance of female rats to cafeteria diet-induced hypertension is abolished by neonatal testosterone treatment. Neonatal androgenization of female rats is known to induce morphological changes in the central nervous system and to irreversibly influence behavior (Simon and Whalen, 1987). In perinatal life, sex steroid hormones regulate neuronal differentiation and synaptic plasticity, thus contributing to the maturation and the sexual dimorphism of the catecholaminergic system in hypothalamus (Simerly, 1989; Siddiqui and Shah, 1997). Moreover, neonatal androgenization of female rats induces male development of the hypothalamic monoamine system (Simerly, 1989; Siddiqui and Shah, 1997). Therefore, the natural androgenization of male pups may induce an irreversible susceptibility to inappropriate responses to the cafeteria diet that lead to hypertension, i.e., hypothalamic over-expression of TH and _2B-adrenoceptor genes, together with a blockade of the adaptive change in _2A-adrenoceptor gene expression. Our present observations in androgenized female rats provide strong support for this hypothesis. Another explanation for our results could be the central implication of estrogens. Indeed, estrogens act centrally to enhance the baroreflex sensitivity (Saleh and Connell, 2000), which inhibits noradrenergic mechanisms in the posterior hypothalamus (Kawasaki et al., 1991). Thus, estrogens may have a protective effect against elevation in blood pressure. Moreover, sex differences in estrogen receptors have been reported in hypothalamus, with higher mRNA levels in female rats (Lauber et al., 1991). This difference may be due to the neonatal peak of testosterone in male pups, which is aromatized in estrogen, and results in decreased central estrogen receptors (Bakker et al., 1997). Therefore, while estradiol and testosterone plasma levels seem to be similar between adult testosterone-treated and intact female rats (Nilsson et al., 1998), neonatal testosterone imprinting of females could lead to altered estrogen receptors, thus reducing the protective effect of estrogens. Further studies on estrogen receptor status in cafeteria diet-fed rats are currently in progress to validate this attractive hypothesis.

In conclusion, our results show that neonatal androgenization of central structure appears to be a determinant factor in the development of cafeteria diet-induced hypertension. Hypertension in male and testosterone-treated female rats is associated to hypothalamic over-expression of TH and _2B-adrenoceptor genes. In contrast, the up-regulation of the _2A-adrenoceptor gene expression in normotensive female cafeteria diet-fed rats could be an adaptive response to this diet that may prevent the onset of hypertension. Conversely, the absence of this adaptive regulation in male and testosterone-treated female cafeteria-fed rats could contribute to the development of elevated blood pressure.