0022-3565/02/3022-525–531$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 302:525–531, 2002
Vol. 302, No. 2
4881/995640
Printed in U.S.A.
Gender Differences in Hypothalamic Tyrosine Hydroxylase and
␣2-Adrenoceptor Subtype Gene Expression in Cafeteria DietInduced Hypertension and Consequences of Neonatal
Androgenization
CHARLES PLUT, CATHERINE RIBIERE, YVES GIUDICELLI, and JEAN-PIERRE DAUSSE
Department of Biochemistry and Molecular Biology, Faculté de Médecine de Paris-Ouest, Université René Descartes, Paris, France
Received December 26, 2001; accepted April 2, 2002
This article is available online at http://jpet.aspetjournals.org
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
This work was supported by grants from the University René Descartes.
over-expression was also induced, but only in male cafeteriafed rats. If one assumes that the up-regulations in TH and
␣2B-adrenoceptor gene expression are indicative of increased
sympathetic nervous activity, then, these altered gene expressions could be responsible for the maintenance of high blood
pressure in male and testosterone-treated female cafeteria-fed
rats. Conversely, in intact females, the absence of these overexpressions and the up-regulation of the ␣2A-adrenoceptor
gene expression could reflect an adaptive response to the diet
and, consequently, could be protective against cafeteria dietinduced hypertension. Moreover, neonatal testosterone imprinting in females could have induced an irreversible android
susceptibility to the cafeteria diet, leading to the onset of hypertension.
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 ␣2Badrenoceptor 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 ␣2adrenoceptor 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). Alto-
ABBREVIATIONS: TH, tyrosine hydroxylase; PCR, polymerase chain reaction; bp, base pair(s); RT-PCR, reverse transcription-polymerase chain
reaction.
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ABSTRACT
This study investigated the incidence of cafeteria-diet induced
hypertension on hypothalamic tyrosine hydroxylase (TH) and
␣2-adrenoceptor subtype gene expression in male, female, and
neonatally testosterone-imprinted female rats. After 10 weeks
of cafeteria diet, all these rats were hyperleptinemic. In contrast, males and testosterone-treated females developed hypertension, whereas intact females remained normotensive. In
these rats, cafeteria diet up-regulated TH gene expression only
in males and testosterone-treated females. On the other hand,
cafeteria diet differentially affected hypothalamic gene expression of ␣2-adrenoceptor subtypes. In fact, this diet increased
␣2A-adrenoceptor mRNA levels only in intact normotensive females. In contrast, gene expression of the ␣2B-adrenoceptor
was up-regulated only in male and testosterone-treated female
cafeteria-fed rats. Furthermore, an ␣2C-adrenoceptor gene
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Plut et al.
Experimental Procedures
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 ␮l, 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 ␮g) 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 ␮l final volume) was carried
out with 10 ␮l 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 ␮Ci 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 ␣2Aand ␣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
TABLE 1
Nucleotide sequences of primers used for PCR amplification
Locus
Primers
Annealing
Temperature
References
280
53°C
Coatmellec-Taglioni et al., 2002
1123
56°C
Yu et al., 1996
311
57°C
Chan et al., 1997
407
57°C
Coatmellec-Taglioni et al., 2002
426
57°C
Chan et al., 1997
Size
bp
␤-Actin
TH
␣2A
␣2B
␣2C
5⬘-TACAACCTCCTTGCAGCTCC-3⬘
5⬘-ACAATGCCGTGTTCAATGG-3⬘
5⬘-GGGATGGGAATGCTGTTCTCAAC-3⬘
5⬘-CGAGAGGCATAGTTCCTGAGCTT-3⬘
5⬘-GCGCCCCAGAACCTCTTCCT-3⬘
5⬘-AGTGGCGGGAAGGAGATGAC-3⬘
5⬘-AGCATCGGATCTTTTTTTGC-3⬘
5⬘-GTTTGGGGTTCACATTCTTC-3⬘
5⬘-CTGGCAGCCGTGGTGGGTTT-3⬘
5⬘-GTCGGGCCGGCGGTAGAAAG-3⬘
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gether, 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.
TH and ␣2-Adrenoceptors in Cafeteria-Fed Rats
527
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
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 testosteronetreated 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). 多多, P ⬍ 0.01; and 多多多, P ⬍ 0.001 for
the comparison between controls and cafeteria-fed rats.
Discussion
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.
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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, testosteronetreated 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 ␣2Cadrenoceptor mRNA levels in male rats but no significant
changes in both groups of females (Fig. 4, bottom).
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Plut et al.
TABLE 2
Physiological parameters of cafeteria-fed rats
Data are means ⫾ S.E.M. Fat mass is mesenteric, perirenal, epididymal/parametrial, and subcutaneous white adipose tissue.
Males
Blood pressure (mm Hg)
Body weight (g)
Fat mass/kg of body mass
Plasma leptin (ng/ml)
Females
Females T-Treated
Control
Cafeteria
Control
Cafeteria
Control
Cafeteria
127 ⫾ 1.2
561 ⫾ 13
58 ⫾ 3.5
6.4 ⫾ 0.5
156 ⫾ 1.7***
628 ⫾ 23*
87 ⫾ 7.8**
12.9 ⫾ 1.3*
132 ⫾ 1
318 ⫾ 6¶¶¶
70 ⫾ 3
5.7 ⫾ 0.8
131 ⫾ 2
386 ⫾ 37*,†††
107 ⫾ 12**
16.4 ⫾ 4*
128 ⫾ 3.7
308 ⫾ 7¶¶¶
47 ⫾ 3
3.3 ⫾ 0.4
169 ⫾ 5.3***,††
400 ⫾ 6*,†††
89 ⫾ 7**
14.2 ⫾ 2.5**
* P ⬍ 0.05; ** P ⬍ 0.01; *** P ⬍ 0.001 cafeteria versus control rats.
††
P ⬍ 0.01, ††† P ⬍ 0.001 versus cafeteria males.
¶¶¶
P ⬍ 0.001 versus control males.
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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 testosteronetreated females (n ⫽ 4). 多, P ⬍ 0.05 for the comparison between controls
and cafeteria-fed rats.
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 phosphor-
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 testosteronetreated females (n ⫽ 4). 多, P ⬍ 0.05 for the comparison between controls
and cafeteria-fed rats.
ylation 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 hypera-
TH and ␣2-Adrenoceptors in Cafeteria-Fed Rats
drenergic 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 ani-
mals. 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 ␣2Badrenoceptor 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 dietfed rats. On the other hand, the up-regulation of the ␣2Aadrenoceptor 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 ␣2Aadrenoceptor 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 ␣2autoreceptors is predominantly mediated by presynaptic ␣2Aadrenoceptors 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
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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 testosteronetreated females (n ⫽ 4). 多多, P ⬍ 0.01 for the comparison between controls
and cafeteria-fed rats.
529
530
Plut et al.
␣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.
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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 ␣2adrenoceptor 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 ␣2Badrenoceptor genes. In contrast, the up-regulation of the
TH and ␣2-Adrenoceptors in Cafeteria-Fed Rats
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Address correspondence to: Dr. Jean-Pierre Dausse, Department of Biochemistry and Molecular Biology, UFR Biomedicale, 45 rue des Saints-Pères,
75270 Paris cedex 06, France. E-mail: jean-pierre.dausse@paris-ouest.
univ-paris5.fr
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