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Rockefeller University, New York, New York 10021; and the School of International Health, University of Tokyo (A.A.), Tokyo, Japan
Address all correspondence and requests for reprints to: Dr. Sarah F. Leibowitz, Rockefeller University, 1230 York Avenue, New York, New York 10021. E-mail: leibow{at}rockvax.rockefeller.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Through pharmacological and biochemical studies in male rats consuming macronutrient diets, these two peptides have been differentially linked to specific macronutrients (1). GAL injections stimulate the ingestion of fat and carbohydrate (5, 6), whereas NPY preferentially increases the intake of carbohydrate (6, 7). Furthermore, measurements of endogenous GAL reveal a close association between peptide gene expression and a rat’s ingestion specifically of fat (8), whereas endogenous NPY levels are higher in rats that prefer carbohydrate (9). These peptides, through their effects on eating behavior, metabolism, and hormone secretion, are believed to be involved in the regulation of body weight (1, 2, 3, 10).
Essentially all of these studies have been performed in male rats. Little is known in female animals about the roles of these peptides in controlling energy balance. Both GAL and NPY have an important function in reproduction, affecting gonadal hormone release and mating behavior (11, 12, 13, 14). Whereas reproduction is very sensitive to the nutritional status of the female (10), and gonadal steroids have potent effects on peptide activity in the brain (11, 13), there are no studies that have directly tested the relationship of these peptides to female patterns of food intake and weight regulation. Greater understanding of this issue may help to explain behavioral and metabolic shifts across the menstrual cycle (15, 16) and the higher risk of obesity for women in certain populations (17). The female steroids have long been known to affect nutrient intake and metabolism (10). However, their precise function and mechanism of action remain to be determined.
This investigation studied female Sprague-Dawley rats in relation to the estrous cycle. Measurements of both peptides, GAL and NPY, as well as the gonadal steroids were made. To relate these steroids and peptides to eating behavior, measurements of food intake, body weight, and body fat were also recorded, and animals with differential preferences for specific nutrients or differential body weights were compared. The results, presented in preliminary form (18), demonstrate marked changes in the peptides and steroids across the estrous cycle, accompanied by changes in eating behavior. Further, in animals showing a stronger preference for dietary fat or greater body adiposity, distinct differences in gonadal steroids and brain peptides are detected.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three groups of female rats were studied at each of the four stages of the estrous cycle. In the first two groups, the animals were maintained on either Standard Purina lab chow (Ralston-Purina, St. Louis, MO; n = 6–7/stage) or macronutrient diets (n = 7–9/stage), and peptide levels were examined using RIA. The third group was fed lab chow (n = 4–5/stage), and their brains were analyzed using immunohistochemistry.
Diets
The animals in the free-feeding, self-selection paradigm were
provided with separate sources of protein, carbohydrate, and fat as
described previously (5, 9). The protein diet (3.7 Kcal/g) consisted of
97% casein (Bioserv, Frenchtown, NJ) mixed with 4% minerals (USP XIV
Salt Mixture Briggs, ICN Pharmaceuticals, Costa Mesa, CA), 2.97%
vitamins (Vitamin Diet Fortification Mixture, ICN Pharmaceuticals), and
0.03% cysteine (L-cysteine hydrochloride, ICN
Pharmaceuticals). The carbohydrate diet (3.7 Kcal/g) was composed of
28% dextrin, 28% corn starch (ICN Pharmaceuticals), and 37% sucrose
(Domino Sugar Co., Cateret, NJ) mixed with 4% minerals and 3%
vitamins. The fat diet (7.7 Kcal/g) consisted of 86% lard (Armour,
Kankakee, IL) mixed with 8% minerals and 6% vitamins. These diets
were placed in separate glass jars at the front of the cage, with their
placement changed daily to prevent position preferences. All rats were
maintained ad libitum on food and water for a period of 4
weeks.
Test procedures
Measurements of food intake were taken daily for a 4-week
period, with data available for each rat at each stage. For all
experiments, rats were killed around the time of dark onset, and blood
was collected for serum analysis of circulating gonadal hormones.
Unilateral body fat from two regions (inguinal and retroperitoneal) as
well as the mesenteric fat pad were collected at the time the rats were
killed and weighed. Total fat pad weights were recorded.
Hormone determinations
Serum LH was determined by RIA using reagents provided by the
National Hormone and Pituitary Program (Baltimore, MD). Serum LH values
are expressed as nanograms per ml rLH NIDDK RP-3 reference standard
(19). Serum PROG levels were assayed with a commercially available kit
(ICN Biomedicals). This assay has a sensitivity of 0.7 ng/ml and an
ED50 of 3.80 ng/ml. The inter- and intraassay coefficients
of variation are 7% and 4%, respectively. 17ß-Estradiol
(E2) was measured in ethyl acetate-hexane (3:2)-extracted
serum using commercially available reagents (Diagnostic Products Corp.,
Los Angeles, CA). This assay has a sensitivity of 8.0 pg/ml and an
ED50 of 170 pg/ml. The inter- and intraassay coefficients
of variation are 8% and 7%, respectively.
RIA (GAL)
Rats were rapidly decapitated, and brains were rapidly removed
and frozen on dry ice for subsequent analysis. Samples micropunched
from nine hypothalamic areas, as described previously (9), were
expelled into 2.0 M acetic acid, and GAL-like
immunoreactivity was measured as previously described (20, 21), using
polyclonal antisera generated in rabbits to a synthetic rat GAL and rat
125I-labeled GAL (Peninsula Laboratories, Belmont, CA). The
samples were reconstituted in assay buffer, and the primary antibody
was diluted in buffer containing normal rabbit serum (1:75,000 and
0.5% final concentration, respectively). Buffer, antibody, samples, or
synthetic rat GAL standards were added at the set-up and incubated for
72 h at 4 C. The radiolabeled GAL was then added, and incubation
was continued for 24 h. Phase separation was achieved by the
addition of goat antirabbit 
-globulin. The assay has a sensitivity
of 4 pg, an ED50 of 55 pg, and intra- and interassay
coefficients of variation of 7% and 18%, respectively.
RIA (NPY)
Rats were rapidly decapitated, and brains were rapidly removed
and frozen on dry ice for subsequent analysis. NPY-like
immunoreactivity in the microdissected tissues was measured by RIA, as
described previously with minor modifications (22). Briefly, the assay
buffer was 0.05 M phosphate buffer (pH 7.4), 0.1% sodium
azide, and 0.25% BSA. Porcine NPY (Peninsula) was used as the
standard. Antiserum for NPY was supplied by Dr. M. R. Brown,
University of California (San Diego, CA). The antiserum (100 µl), at
a final dilution of 1:240,000, and the standards or reconstituted
samples (200 µl in assay buffer) were preincubated for 24 h at 4
C. Then, 100 µl tracer ([125I]NPY labeled with
Bolton-Hunter reagent, NEX222, DuPont Chemicals, Boston, MA) were added
and incubated for an additional 24 h. The free and antibody-bound
NPY were separated by adding 500 µl charcoal (2.5%) and dextran-70
(0.25%) solution. Bound fraction was measured in a -counter. The
final dilution of antibody was chosen to achieve a total binding of
[125I]NPY at 40–45%. The assay sensitivity was 10
pg/tube, and the decrease of 50% in the bound activity was obtained
for 50 pg/tube. Nonspecific binding for the assay buffer was less than
9.0%, and intra- and interassay coefficients of variation were 5.0%
and 9.4%, respectively.
Immunohistochemistry
For immunohistochemistry (23), all rats were anesthetized with
an overdose of Metofane and perfused via the ascending aorta with 200
ml 0.9% NaCl followed by 400 ml 4% paraformaldehyde in 0.1
M phosphate buffer (pH 7.4). Brains were removed, postfixed
in the same phosphate-buffered solution overnight at 4 C, and stored in
a 30% sucrose-phosphate buffer containing 0.01% sodium azide (pH
7.4) at 4 C for 48 h. The brains were then frozen at -80 C until
the day of use.
For the experiments, the brains were cut into 30-µm thick sections with a cryostat. A consistent angle of cut was maintained by examining the shape of the third ventricle. Brains within a given experiment were always processed at the same time to maintain stringent tissue preparation and staining conditions. Incubation then occurred with normal goat serum (dilution 1:10 in PBS with 0.5% Triton X-100) for 30 min. The tissues were transferred into either a NPY or a GAL primary antibody (NPY dilution, 1:12,500; supplied by Dr. Marvin R. Brown, University of California, San Diego; GAL dilution, 1:20,000; supplied by Dr. Steven M. Gabriel, Mt. Sinai School of Medicine, New York, NY) at room temperature for 24–48 h. The tissues were then exposed to secondary antiserum, biotinylated antirabbit IgG (Vectastain Elite Kit, Vector Laboratories, Burlingame, CA), for 1 h. The sections were processed further using standard Vectastain ABC techniques. Staining finally occurred after exposure to 0.05 M Tris-HCl buffer solution with 0.01% 3,3'-diaminobenzidine tetrahydrochloride (Sigma) containing 0.03% H2O2. Between each step, the sections were rinsed twice with 0.1 M PBS (pH 7.4). The sections were mounted on slides, dehydrated with a graded series of ethanol and xylene, and coverslipped.
Quantification of peptide immunoreactivity and statistical
analysis
A digital imaging system, with the help of a rat brain atlas
(24), was used for peptide quantification in brain tissue. Different
hypothalamic and extrahypothalamic areas were examined unilaterally on
four anterior-posterior levels: 1) the medial preoptic area (MPOA;
bregma 0.6 mm, predominantly the middle portion); 2) the anterior
parvocellular part of the paraventricular nucleus (aPVN), the
supraoptic nucleus (SON), and the suprachiasmatic nucleus (bregma 1.4
mm); 3) the lateral magnocellular part vs. the medial
parvocellular part of a more posterior region of the PVN (bregma 1.8
mm); and 4) the arcuate nucleus (ARC), median eminence-external zone
(ME), dorsomedial nucleus (DMN), and central nucleus of the amygdala
(bregma 2.8 mm). The density of cells or fibers in approximately three
sections at the same level was measured for each rat. A Leitz
microscope (Leitz, Rockleigh, NJ) was used with a x4 illumination
objective when focused on the cells and a x10 objective when focused
on the fibers. A video camera connected to an IBM computer with WScan
Array Software (Galai Production Ltd., Migdal Haemek, Israel) was
converted to a digital image, with a gray value ranging from 0–255. To
count the number of black pixels, a threshold was established, above
which pixels were counted. The threshold was the same for all sections
counted.
Data analysis
The animals’ Kcal intake scores were averaged across the 4-week
test period, and the mean scores represent Kcal per 24 h. Body
weight, body weight gain, and body fat are presented for the final week
of measurements. Food intake measurements were analyzed via ANOVA with
repeated measures, whereas measurements of body weight, hormones, and
peptides were performed using a one-way ANOVA, followed by Duncan’s
new multiple range test or Student’s t test when
appropriate. Measures of nutrient intake, body weight, hormones, and
hypothalamic peptide levels were related using a Pearson’s
product-moment correlation. Macronutrient intake data are presented as
Kcal per 24 h as well as a percentage of the total Kcal intake.
For determination of low fat eaters and high fat eaters, nutrient Kcal
intake was averaged across all four stages of the cycle. Based on their
scores for fat preference, the rats were then rank ordered and
separated into two groups consisting of the 40% lowest or the 40%
highest. Similarly, to create low and high body weight subgroups, the
body weight scores during the final week were rank ordered and
separated into two groups consisting of the lowest 40% or the highest
40%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hypothalamic GAL across the estrous cycle
Analyses of these three groups reveal consistent patterns of
peptide levels across the estrous cycle. The RIA measurements of GAL in
the animals receiving lab chow (Fig. 1
, top) or macronutrient diets (Fig. 1, bottom)
showed a peak of GAL levels during the proestrous stage. These analyses
distinguished three specific areas with this pattern. These were the
MPOA, PVN, and ME, where GAL levels are 50–100% higher during
proestrous compared with those during the other stages of the cycle. In
both groups, there was little change in GAL in other hypothalamic areas
examined, including the ARC (Fig. 1) as well as the VMH, DMN, and SON
(data not shown). A close association among the MPOA, PVN, and ME in
their shift across the cycle was reflected by positive correlations
detected between GAL levels in the PVN and ME (r = 0.84;
P < 0.05) and GAL in the MPOA and ME (r = 0.81;
P < 0.05).
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). In the MPOA, a rise in GAL
immunoreactivity during proestrous was seen throughout this area,
predominantly in the fibers, but also in the few visible neurons (see
arrows in Fig. 2, A and B). In the PVN, the aPVN was the
site within this nucleus showing the greatest shift during proestrous
(+30%; P < 0.05; Fig. 2, C and D). This area had a
dense concentration of both fibers and neurons, although only a few
neurons (see arrows) could be seen through the dense fiber
staining. In contrast to the aPVN, the central portion of the PVN,
where fibers were concentrated medially and GAL-containing
magnocellular neurons existed laterally just caudal to the aPVN, showed
only a small, insignificant rise in GAL staining at proestrous (data
not shown). In the ME, the proestrous peak in GAL immunoreactivity was
seen in the external zone (Fig. 2, E and F). As with the RIA, no
changes in GAL fiber density were detected in other areas, including
the ARC and DMN (data not shown).
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) and also from
analyses of NPY immunoreactivity using immunohistochemistry in lab chow
diet subjects (Fig. 4).
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, top) and those
receiving macronutrient diets (Fig. 3, bottom). No change in
NPY was seen in other hypothalamic areas, including the ME (Fig. 3),
DMN, VMH, and suprachiasmatic nucleus (data not shown).
The immunohistochemical analyses (Fig. 4) demonstrated that the
NPY-containing neurons of the ARC that showed increased staining during
proestrous were located in the mediodorsal area of the nucleus (see
arrows), among a dense concentration of fibers. The NPY
terminals of the MPOA and PVN that exhibited a proestrous rise were
concentrated in the medial regions of these nuclei (Fig. 4). The
existence of an NPY projection, from the ARC to the PVN or MPOA, that
impacts on the female cycle during proestrous was supported by a
correlational analysis of the animals receiving macronutrient diets. In
these rats, a positive correlation was detected between NPY levels in
the ARC and peptide in the PVN (r = 0.67; P <
0.05), MPOA (r = 0.69; P < 0.05), or ME (r =
0.72; P < 0.05).
Hormone measurements across the estrous cycle
In each of the three groups, the animals were killed, and
their blood samples were collected before the onset of the dark cycle
when spontaneous feeding initiates. The hormone measurements for the
two lab chow groups, which were combined, and the macronutrient diet
groups are presented in Tables 1 and 2. The results reveal clear,
stage-dependent shifts in the levels of gonadal hormones in the rats.
In the lab chow and macronutrient diet groups, peak LH levels were
detected during proestrous. Whereas E2 showed a small rise
during this stage, PROG increased significantly to levels approximately
30–40% higher in proestrous animals (P < 0.05) than
those seen during the other stages (Tables 1 and 2).
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and 2. In all groups, a significant decline in
total caloric intake was evident during the estrous stage, when body
weight was also lowest. In rats receiving macronutrient diets, this
reduction in food intake during estrous was characterized by a specific
drop in fat ingestion, with no change in carbohydrate or protein intake
(Table 2). Further, when fat intake was calculated as a percentage of
the total intake, a significant enhancement of the rats’ relative
preference for this nutrient was evident during proestrous. This
increase in fat preference was accompanied by a 50% increase in the
size of the inguinal fat pad from diestrous (6 ± 0.5 g) to
the end of proestrous (9 ± 0.6 g; P <
0.05), followed by a decline by the end of estrous. No significant
change was evident in the weight of the other fat pads (data not
shown).
Relationships among measurements of the peptides, steroids, fat
intake, and body fat
This evidence indicates that GAL and NPY, specifically in the PVN,
ARC, MPOA, and ME, increase during proestrous. At this same time, there
occurred a rise in the gonadal steroids in addition to an increase in
the rats’ preference for fat and the weight of their inguinal body fat
pad. The question to be addressed here is whether these temporally
associated phenomena are directly and perhaps functionally related.
Correlational analyses within groups reveal some associations that are at least consistent with this possibility. The strongest correlations are detected for hypothalamic levels of GAL. For example, this peptide in rats receiving lab chow was positively related to circulating levels of PROG, specifically in the MPOA (r = 0.76; P < 0.05), PVN (r = 0.88; P < 0.05), and ME (r = 0.96; P < 0.05), whereas PVN GAL was also correlated with total Kcal intake (r = 0.81; P < 0.05). In the diet choice group, the importance of a specific nutrient in this relationship was supported by positive correlations, specifically during proestrous, between the ingestion of fat, but not carbohydrate or protein, and GAL in the PVN (r = 0.67; P < 0.05), ME (r = 0.69; P < 0.05), and, insignificantly, MPOA (r = 0.46; P = NS). Further, an association with body fat was reflected in its positive correlation with fat ingestion (r = 0.80; P < 0.01), PROG (r = 0.92; P < 0.01), and PVN GAL (r = 0.73; P < 0.05).
With regard to NPY, positive associations in the rats receiving lab chow were also seen. Levels of NPY in the ARC were correlated with total Kcal intake (r = 0.75; P < 0.05), whereas NPY in the MPOA was positively related to body fat pad weights (r = 0.96; P < 0.01). In the macronutrient diet paradigm, rats showed a correlation between circulating levels of LH and NPY levels in the MPOA (r = 0.75; P < 0.05) and the PVN (r = 0.92; P < 0.01).
Differential traits in high fat eaters and in relation to body
fat
These associations suggested by the correlational analyses
of the different measures are further strengthened by examination of
animals differentiated on the basis of their fat preference or body
weight. In rats maintained on the macronutrient diets, a range of
preference for dietary fat was evident, from 25–62% of the total
diet. Therefore, the animals could be readily separated into two
subgroups, consisting of high fat eaters (HF; n = 15; upper 40%
of the total group) with more than 40% dietary fat and high
carbohydrate eaters (HC; n = 15; lower 40% of the total group)
with less than 35% dietary fat. These subgroups show very different
patterns of eating behavior, steroids, and peptide levels (Fig. 5).
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), consistent with the increase in fat preference
demonstrated for the total group. The HF eaters, however, fail to
demonstrate this stage-dependent shift, showing a generally high
baseline of fat intake at all stages of the cycle (Fig. 5). This
increased fat intake along with a higher body weight (+9%;
P < 0.05) were accompanied by significant changes in
hormone levels. The HF eaters compared with the HC eaters had
significantly higher levels (
50%) of both E2 and PROG
(P < 0.05).
The hypothalamic peptides also differed between these two subgroups
(Fig. 5), specifically in the same areas that exhibited a peptide shift
across the estrous cycle. In the HF eaters compared with the HC eaters,
GAL levels were significantly higher in the MPOA, PVN, and ME, but
showed no difference in the ARC, VMH, DMN, or SON (data not shown).
Measurements of NPY also revealed significantly higher peptide levels
in the MPOA of HF eaters (Fig. 5). This was in contrast to the ARC and
PVN, where NPY levels were reliably lower in the HF eaters.
Whereas fat intake and body fat were positively related, as
indicated above, subgrouping these same animals during macronutrient
diets according to their body weight generated a somewhat different set
of animals. This analysis confirmed the importance specifically of the
MPOA and its peptide level in relation to body fat (Fig. 6). Rats subgrouped as high body weight,
compared with low body weight subjects (n = 15/group), had 50%
more body fat and consumed approximately 5 more Kcal fat/day. These
traits were accompanied by significantly higher levels of PROG, but not
E2. In addition, these heavier subjects had higher levels
of the peptides. This was seen for both GAL (+30%; P
< 0.05) and NPY (+25%; P < 0.05) specifically in the
MPOA (Fig. 6), but not in any other hypothalamic area examined (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptide levels and circulating steroids across the estrous
cycle
Prior studies have demonstrated cycle-related changes in the
peptides within specific areas, with peaks during proestrous (11, 13).
This has been observed with measurements of GAL gene expression and
peptide levels in the MPOA and GAL peptide in the ME (25, 26, 27). For NPY,
in contrast, a peak during proestrous is evident in the ARC for both
messenger RNA and peptide levels and in the MPOA and ME for NPY peptide
levels (28, 29). The present findings are consistent with these
patterns, as revealed through measurements of peptide levels via RIA in
micropunched areas as well as peptide immunoreactivity using
immunohistochemistry.
A specific hypothalamic area receiving little attention in these studies, however, is the PVN. In this nucleus, the present experiments demonstrate a similar increase in peptide levels during proestrous. For GAL, this shift occurs specifically in the aPVN, where dense GAL-containing neurons as well as fibers exist, but not the central portion of the PVN, which also has a group of GAL neurons. This is in contrast to NPY, for which the proestrous rise occurs specifically in the medial area of the PVN, where NPY-containing fibers are particularly dense.
As expected based on published studies (13), levels of the gonadal steroids, E2 and PROG, and the GnRH, LH, also rise during proestrous. Correlational analyses consistent with these temporal patterns reveal significant associations between the gonadal hormones and brain peptides. In particular, a consistent, positive relation is seen between circulating PROG and GAL levels in the MPOA, PVN, and ME. Interestingly, this association is evident primarily during the proestrous period, when both the steroid and peptide are most active and are likely to functionally interact. Whereas E2 is known to stimulate GAL in several areas, including the MPOA, PVN, and ME, in addition to the anterior pituitary (20, 26, 30, 31, 32), PROG in E2-primed rats produces a further enhancement of GAL in the hypothalamus and pituitary (33) and specifically the MPOA (34). Gonadal steroid receptors and GAL are concentrated and possibly coexist in these areas (11).
A clear relationship between NPY and the gonadal steroids is not as evident in the correlational analyses of this study. This may be due to the fact that E2 and PROG have both inhibitory and stimulatory effects, acting on local steroid receptors, on the NPY projections in different brain areas (13, 35, 36, 37, 38). What is observed here, however, is a positive relation between circulating levels of LH and NPY levels in both the MPOA and PVN. This relationship may reflect a stimulatory effect of NPY on LH secretion that peaks at the start of proestrous (13, 28).
Patterns of eating behavior across the estrous cycle
There is considerable evidence for a shift in eating behavior
across the female cycle, showing a decline during the estrous phase
(10, 39). This is confirmed here by a significant reduction in total
caloric intake during this period in all groups and specifically a
decline in fat ingestion in the diet choice group. This effect may be
attributed in part to the sharp decline during estrous in both GAL and
NPY in the PVN. This nucleus or its immediate area is a primary site
for stimulating feeding with local injections of these peptides, as
demonstrated in male rats (1, 5). It is also responsive to GAL and NPY
receptor antagonists or antisense oligonucleotides that reduce feeding
behavior and body weight (8, 22). Whereas the responsiveness of the
MPOA to peptide injections has yet to be systematically tested in
females, there is some evidence in males that GAL and NPY in this area
may have some effect on feeding (40) as well as energy expenditure
(1).
Of particular interest are the behavioral data obtained from the animals receiving the macronutrient diets. These results reveal a significant shift in the rats’ nutrient preference across the estrous cycle, involving a significant increase in preference for dietary fat during the proestrous stage. This pattern was first suggested by an earlier study showing a rise in fat preference subsequent to estrous, presumably during the proestrous period (41), although another study reported little change (42). From the present results, it is of interest that this rise in dietary fat is followed by an increase in inguinal fat pad weights before the start of estrous. Whereas there are apparently no prior studies that have measured body fat pad weights across the estrous cycle in rats, measurements of body weight reveal the greatest weight gain during diestrous and proestrous (10, 43).
Hypothalamic peptides in relation to macronutrient intake
Investigations in male rats have demonstrated a strong
relationship between fat ingestion and GAL specifically in the PVN and
ME (1). This was first demonstrated through measurements of GAL levels
in micropunched areas (8) and has been confirmed more recently using
in situ hybridization and immunohistochemistry (44). In
these investigations, GAL messenger RNA in the aPVN and peptide-ir in
both the aPVN and external zone of the ME are closely correlated with
the amount of fat ingested, which, in turn, is linked to body fat. This
relationship in male rats is not detected in any other hypothalamic
area, including the MPOA.
In the female rats of this report, GAL is similarly related to fat ingestion. As in male rats, the aPVN and ME are distinguished in this manner. This is reflected by correlational analyses showing a positive relation between fat intake and PVN or ME GAL and also by the similar temporal shift in both GAL and fat ingestion across the estrous cycle, with a peak during proestrous. Of particular note, however, is the evidence obtained for the MPOA; this area is similarly related to fat ingestion in females, but apparently not in males (8, 34). Levels of the peptide in this area are positively correlated with fat consumption, and they exhibit a rise during proestrous when dietary fat is naturally preferred.
In the male rat, distinct differences between NPY and GAL are evident in terms of their relation to nutrient ingestion. In contrast to GAL, NPY has been associated with dietary carbohydrate (1, 2, 9), with NPY levels in the ARC and PVN invariably higher in rats that consume more of this nutrient (9, 45, 46). Whereas these areas exhibit a similar pattern in the female rat, NPY in the MPOA responds differently. In contrast to the male rat, in which NPY and GAL in this area show little change in relation to dietary nutrients (8, 9), female rats have significantly higher NPY levels in the MPOA of the HF group. Thus, for both NPY and GAL, peptide levels in this area appear to be specifically linked to fat ingestion. There are no studies that have directly compared males and females in terms of their responses to peptide injections. In one report, however, chronic hypothalamic injections of NPY in female rats stimulate daily consumption of both carbohydrate and fat (47).
Circulating steroids and hypothalamic peptides in relation to
nutrient ingestion and body fat
The additional involvement of the steroids, in particular
PROG, in this relationship between hypothalamic peptides and fat intake
is suggested by several findings in this study. The temporal
associations between these measurements across the estrous cycle are
evident, as are the positive correlations between PROG and GAL.
Moreover, PROG and fat ingestion themselves are positively related,
with the HF group exhibiting significantly higher levels of the
circulating steroid.
A functional relationship between this steroid and the peptides is supported by the stimulatory effect of PROG injections in E2-primed rats on GAL and NPY gene expression in the MPOA (11, 13). This suggests that PROG may mediate the rise in GAL and NPY observed in HF eaters. Support for this proposal is obtained from the additional finding that PROG has a stimulatory effect on food intake in E2-primed OVX rats (10, 48). This indicates that this steroid, through a stimulation of peptide production, may have a functional role in controlling eating behavior across the reproductive cycle. This interaction is exaggerated in animals that overconsume a high fat diet.
Fat ingestion is known to be strongly correlated with body fat (49), a relationship also found in the present study. Thus, a consequence of this association between dietary fat and peptide levels, both GAL and NPY, in the MPOA may be an increase in body fat deposition. In fact, comparisons between rats of differential body weights show the heavier subjects, with greater body fat, to have higher circulating levels of PROG, even though their fat ingestion is only slightly higher than that of the leaner rats. These heavier rats also have higher levels of GAL and NPY, specifically in the MPOA, and they exhibit a significant positive correlation between MPOA peptides and body fat. Thus, in addition to the behavioral process of fat ingestion, peptide levels specifically in the MPOA are related to adiposity in female subjects, in contrast to males. The involvement of PROG in this relationship is supported by published evidence showing its stimulatory effect on lipogenesis and body fat and its inhibitory effect on fat oxidation (10, 48, 50). This steroid, through its impact on GAL and NPY projections, may be involved in the normal shift in eating and body weight across the estrous cycle. It may also have a role in the development of obesity in animals consuming a high fat diet.
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Acknowledgments |
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Footnotes |
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Received August 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-methyl-19-nortestosterone is not amplified in male reproductive
tract as is that of testosterone. Endocrinology 130:3677–3683[Abstract]
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 A. Jureus, M. J. Cunningham, D. Li, L. L. Johnson, S. M. Krasnow, D. N. Teklemichael, D. K. Clifton, and R. A. Steiner Distribution and Regulation of Galanin-Like Peptide (GALP) in the Hypothalamus of the Mouse Endocrinology, December 1, 2001; 142(12): 5140 - 5144. [Abstract] [Full Text] [PDF]
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S. H. Russell, C. J. Small, A. R. Kennedy, S. A. Stanley, A. Seth, K. G. Murphy, S. Taheri, M. A. Ghatei, and S. R. Bloom Orexin A Interactions in the Hypothalamo-Pituitary Gonadal Axis Endocrinology, December 1, 2001; 142(12): 5294 - 5302. [Abstract] [Full Text] [PDF]
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 S. R. Inamdar, K. M. Eyster, and E. H. Schlenker Genome and Hormones: Gender Differences in Physiology: Selected Contribution: Estrogen receptor-{alpha} antisense decreases brain estrogen receptor levels and affects ventilation in male and female rats J Appl Physiol, October 1, 2001; 91(4): 1886 - 1892. [Abstract] [Full Text] [PDF]
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 M. DE JONG-BRINK, C. N. REID, C. P. TENSEN, and A. TER MAAT Parasites flicking the NPY gene on the host's switchboard: why NPY? FASEB J, November 1, 1999; 13(14): 1972 - 1984. [Abstract] [Full Text]
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C. Faure-Virelizier, D. Croix, S. Bouret, V. Prevot, S. Reig, J.-C. Beauvillain, and V. Mitchell Effects of Estrous Cyclicity on the Expression of the Galanin Receptor Gal-R1 in the Rat Preoptic Area: A Comparison with the Male Endocrinology, October 1, 1998; 139(10): 4127 - 4139. [Abstract] [Full Text] [PDF]
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