E .E. Otukonyong et al. Brain Research 887 2000 70 –79
71
weight gain [56,60] through a negative feedback loop leptin to restoring the hormonal levels of several reproduc-
involving receptors in the hypothalamus [49]. The recent tive hormones during nutritional distress [1,5]. There is no
demonstration of leptin receptors in the ventromedial previous study to date which reported the effect of leptin in
hypothalamus VMH
[47], arcuate
nucleus ARC
starvation on the level of estrogen, an important hormone [31,43], ovary [25,27], paraventricular and periventricular
whose release precedes the release of LH. This study was nuclei [15], provides the basis for a neuro-endocrine role
designed to examine the possible effects of food depriva- of leptin in the control of anterior pituitary and ovarian
tion and leptin repletion on the plasma levels of estrogen at hormones secretion. Nitric oxide NO has been shown to
different times on proestrus and NOS reactivity in the be positively correlated to increased food intake and body
VMH and ARC. weight [34,35], and regulates thirst and water intake [9,10].
Recent evidence has implicated NO in many actions of leptin leading to the enhanced secretion of reproductive
2. Materials and methods
hormones such as LHRH from the hypothalamus [40], LH and FSH from the adenohypophysis [57,58]. NOS expres-
2.1. Animals and treatments sion and NO generation have been reported in the ovaries
of rats [22,51] and shown to play several roles in ovarian Female Wistar Imamichi rats Hamamatsu, Japan
steroidogenesis [50] and ovulation [45]. All these findings weighing 230–240 g, were housed under controlled tem-
suggest that NO may be involved in the regulation of perature 24618C and photoperiod 14L:10D, lights on
energy metabolism and reproduction. The importance of at 05.00–19.00 h and free access to food Oriental Yeast,
these hypothalamic sites: the ARC and VMH in the Japan and water. Estrous cycles were monitored daily
regulation of metabolism and feeding behavior has been 09.00–10.00 h by vaginal lavages. Female Wistar Im-
well documented [6,14]. For instance, VMH acting as an amichi rats were used in these experiments because of their
important control center for food intake and adiposity, has regular 4-days cycles. Rats were divided into five groups
been shown to sensor blood glucose, blood amino acids for use in five different experiments. All rats were fasted
and also gives efferent signals to suppress food intake and and deprived of food but not water. Food was withdrawn
energy expenditure [7]. Thus, destruction of VMH has from the animals at 09.00 h on diestrus II because this is
been reported to be accompanied by the subsequent when food intake and body weight gain in rats are highest
increase in food intake and decrease in energy expenditure [53,54]. Leptin Mouse recombinant, NHPP-NIDDK,
which results in massive obesity [8,21]. Central injection USA or vehicle was given i.p. to all rats except those fed
of galanin, a feeding-related neuropeptide into the VMH on the 3rd and 4th day at 1000 h and 1700 h. Body weight
increased feeding in rats [24]. VMH also contains both was investigated before and after fasting. The fed animals
NADPH-diaphorase [52] and estrogen binding sites [39]. were allowed free access to food and water ad libitum.
The importance of ARC in the regulation of energy Adequate measures were taken to minimize stress or pain
homeostasis has been confirmed and strengthened by a to the animals.
number of recent studies. For example, coexpression studies indicate that a population of ARC neurons syn-
2.2. Experiment 1 thesizes both agonist for an anabolic neuroendocrine
system neuropeptide Y NPY and an antagonist for a In this experiment, the effect of starvation and leptin
catabolic system agouti-related protein [32]. In addition, repletion on the plasma levels of estrogen 17-b-estradiol
feeding-related neuropeptides
such as
leptin, pro-
was investigated by radio-immunoassay method. All the opiomelanocortin POMC and NPY have been shown to
reagents used were bought commercially from Nacalai coexpress with neurons of the ARC [13,31]. A recent
Tesque Inc., Kyoto, Japan unless otherwise stated. report indicates that damage to ARC produces massive
obesity in birds and mammals [8]. Increased NPY and 2.2.1. Reagents
decreased POMC mRNA in the ARC has been reported in The reagents used in this assay were tracer 2, 4. 6. 16.
fasted animals [26,30,43]. 17-3H E2 NEN, antiserum 17-b-estradiol-6-CMO-BSA
It is now known that undernutrition affects all levels of Biogenesis, New Fields, UK, standard 17-b-E2 powder
the hypothalamic–pituitary–ovarian axis. The sequential in methanol, 0.01 M sodium phosphate buffer pH 7.5 in
secretion of estradiol and progesterone has been reported 0.9 NaCl, 0.1 sodium azide and 0.1 gelatin Difco,
to be disrupted by food deprivation [33]. A substantial Laboratories, Detroit, Michigan, USA, dextran–charcoal
reduction even in the estrogen-binding neurons in the prepared from dextran Wako Pure Chemical Industries,
VMH and in the area lateral to it has also been reported Japan, Norit A in cold buffer, diethylether, aquasol NET,
[28]. Although the exact role of leptin in starvation has not Universal L. S. C and methanol.
been completely elucidated, evidence for its role in the restoration of hormonal levels and reproduction in mam-
2.2.2. Experimental protocols mals is overwhelming. There are several reports linking
Animals were divided into three groups. The first group
72 E
was fed; the second group was fasted and given 0.9 in calculating the level of estradiol. The assay recovery
saline vehicle and the third group received 100 mg leptin rate was 98.7 and the assay sensitivity was 32 pg ml.
as 1 ml. Both the leptin and the vehicle were given i.p. on The inter- and intra-assay coefficients of variation were 4
the 3rd and 4th last day of the fasting treatment twice and 3, respectively.
daily, i.e., at 10.00 h and 17.00 h. Fasting which started at diestrus was terminated at diestrus covering a one 4-day
2.3. Experiment 2 estrous cycle. Food was returned to all the animals at the
end of the treatments i.e., 16 h after the last dose of leptin The plasma levels of leptin following fasting and leptin
or vehicle. On the following day, proestrus trunk blood treatment were also investigated. The plasma used for E2
was collected into heparinized tubes by neck decapitation RIA were also used to measure leptin levels. This was to
with guillotine at 10.00 h n55, 14.00 h n55, 18.00 h enable us to know how high the serum leptin rose
n55, and at 22.00 h n55. The blood was immediately following injections of leptin. Plasma leptin levels were
centrifuged at 4000 rpm for 4 min at 48C and the plasma determined by using a specific rat leptin RIA kit purchased
stored at 2208C until assayed for estradiol E2. from Linco Research St. Louis, MO, USA. The sensitivi-
ty of the assay was 0.5 ng ml. The intra- and inter-assay coefficients of variation were 5 and 6, respectively.
2.2.3. Estrogen assay procedures Plasma estrogen was assayed by the method of Albrecht
2.4. Experiment 3 et al. [4] with some modifications. Briefly, the working
tracer for RIA was prepared by drying 10 ml of stock This experiment examined the effect of fasting and
tracer in nitrogen gas and adding 8 ml of phosphate buffer leptin repletion on nitric oxide synthase NOS reactivity
to the dried up tube. This was stabilized on ice at a final in the ventromedial VMH, arcuate ARC nuclei using
concentration of 10,000 dpm 100 ml and used for RIA. NADPH-d histochemistry, a marker for neurons expressing
For the assay recovery rate, 1 ml of the working tracer was NOS enzyme. Rats were divided into three groups of five
taken and 9 ml of phosphate buffer added. This was rats each n 55. The first group was fed, the second group
stabilized on ice at a final concentration of 1000 dpm 100 was fasted and given vehicle, the third was fasted and
ml 310 dilution. received leptin. The treatments procedure and leptin dosing
are as described in Experiment 1. Animals in the three 2.2.4. Extraction of E2
groups were killed in random order between 09.00 and The 17-b-estradiol was extracted by diethyl ether. Into a
13.00 h. glass tube, 200 ml of plasma, 500 ml of buffer and 100 ml
of tracer 1000 dpm 100 ml, and 4 ml of ethyl ether were 2.4.1. Tissue preparation
added, vortexed and allowed to stand in a freezer for 20 Under pentobarbital anesthesia, animals were transcar-
min. The ethyl ether layer was carefully taken to culture dially perfused with 100 ml of ice-cold buffer 0.1 M
glass tubes, dried in nitrogen gas at 378C. One milliter of phosphate buffer, PB and 200 ml of ice-cold 4 parafor-
methanol was added to the dried up tubes and then maldehyde PFA in 0.2 M PB. The brain tissues were
vortexed. One hundred microliter of methanol was added quickly removed and postfixed in 4 PFA at 48C and then
to each vial bottle in 5 ml aquasol and counted for 5 min. cryoprotected in 30 sucrose solution for 2 days at 48C.
The assay recovery rate was calculated using the formula: Hypothalamic coronal sections 50 mm were cut on a
[C. sample 2 C. Bg 3 10 T.C. 2 C. Bg] 3 100 cryostat Reichert–Jung, Cryocut 1800, Finetec Scientific
Instruments, Tokyo, Japan at 2188C. The VMH and ARC Where: C. sample5sample value; C. Bg.5background
levels were identified according to their stereotaxic coordi- value; T.C.5Total count
nates in the rat brain atlas [38]. The sections were placed in 0.1 M PB after which they were placed to wash on an
2.2.5. E2 RIA procedure electronic shaker Taitec Instruments, Saitama, Japan for
Into the dried up tubes, and tubes for the standards, 100 30 min.
ml of buffer, 100 ml of tracer and 100 ml of antiserum 1:4000 were added and incubated in a cold room
2.4.2. NADPH-d histochemistry overnight at 48C. After which, cold buffer and dextran–
The sections were processed for NADPH-d histo- charcoal solution were added on ice bath, the contents
chemistry by incubating in a medium containing 10 mg vortexed and allowed to stand for 5–10 min. The tubes
b-NADPH-d Sigma Chemical Company, St. Louis, USA, were then centrifuged at 2000 rpm for 10 min at 48C and
1 mg nitroblue tetrazolium Wako Pure Chemical Indus- the upper layer 0.85 ml translated to vial bottles in 5 ml
tries, Tokyo, Japan, 0.03 Triton X-100 Nacalai Tesque aquasol. Each vial was counted for 5 min. The dextran-
Inc., Kyoto, Japan in 10 ml 0.02 MPB for 1 h. The coated charcoal suspension method separates bound and
reaction was stopped by washing in 0.01 M PB. Sections free tracer. A logit-log dose regression equation was used
were mounted on gelatin-coated slides, cleared in xylene
E .E. Otukonyong et al. Brain Research 887 2000 70 –79
73
and coverslipped using Eukitt reagent O. Kindler, MA-2091, Japan. The stored images were analyzed for
Feiburg, Germany and examined under a light micro- cell count and intensity by selecting appropriate menus
scope. NADPH-d-positive cells were counted per section. such as slice density under the ‘option’ menu at 256 gray
The counting and staining intensity quantification was levels pixel, the ‘tools’ menu to map out the area to be
done by using computer software NIH image version 1.58. counted and the ‘lut’ menu to set the intensity threshold
Some cells were counted manually at 320 objective using an NIH Image, version 1.58 software with a mini-
magnification. mum particle size setting of 10 pixels and standard
threshold. The cells were counted regardless of the intensi- 2.5. Experiment 4
ty of staining since the weaker intensity observed in some sections may possibly have resulted from fixation. Some
In this experiment, we investigated the effect of fasting cells were counted manually at 320 objective magnifica-
treatment and leptin repletion on ovulation. This was to tion. The intensity values which represent the product of
know whether leptin treatment was effective in reversing signals that take both the numbers and the intensity of
the starvation-induced inhibition of estradiol secretion, and staining into account are presented in arbitrary units.
which by implication and extension infers that probably the secretion of LH ovulatory hormone may have
2.8. Statistical analysis occurred in the afternoon of proestrus. The treatment
procedure and the leptin dosing is as described in Experi- At least six sections were counted for each animal and
ment 1. Ovariectomy was performed under light ethyl five sections with the highest numbers of NADPH-d-
ether in the morning of estrus. The excised ovary and the positive cells were used for statistical analysis. Data are
isolated oviduct were covered with drops of saline 0.9 presented
as means6standard
error of
the mean
and coverslipped. The oviduct portion of the ovary was means6S.E.M.. NADPH-d-positive cells, staining in-
examined under a light microscope for an extruded ovum. tensity, estrogen and leptin data were analyzed using the
Mann–Whitney U-test. Incidence of ovulation was ana- 2.6. Experiment 5
lyzed by Fisher’s exact probability test. Comparison between groups for body weight change, food intake and
This experiment was designed to investigate the effect water intake was by Student’s t-test.
of leptin treatment on dependable variables such as food intake, water intake and body weight change. This was to
be used as a strong independent measure of ascertaining
3. Results
