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Division of Neuroscience (R.S.B., M.S.S.), Oregon Regional Primate Research Center, Oregon Health Sciences University, Beaverton, Oregon 97006; and Rowett Research Institute (S.E.M., P.T.), Aberdeen AB21 9SB, Scotland, United Kingdom
Address all correspondence and requests for reprints to: M. Susan Smith, Ph.D., Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: smithsu{at}ohsu.edu
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Abstract |
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Introduction |
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Lactation is also characterized by an inhibition of reproductive cyclicity that most likely results from the suppression of pulsatile LH secretion, and thus inadequate ovarian stimulation. The suppression of LH secretion during lactation occurs independently of the action of ovarian steroids, since pulsatile LH secretion is suppressed in both ovarian intact and ovariectomized (OVX) lactators (12, 13). The mechanisms by which the suckling stimulus and/or the changes in energy balance induced by the metabolic drain of milk production interact at the hypothalamic-pituitary axis to suppress LH secretion are currently unknown.
It is possible that the changes in energy balance and in various metabolic signals may be related to the suppression of pulsatile LH secretion during lactation. A likely candidate that could link changes in energy balance with changes in reproductive function is leptin. Leptin, the product of the ob gene is produced primarily by adipose tissue and is secreted from adipocytes in the fed state; it has been termed the "satiety" factor (14). Absence of leptin is associated with obesity, whereas administration of leptin to the ob/ob mouse, which lacks functional leptin and the fa/fa rat has been shown to reduce food intake and lower body weight (15, 16, 17, 18, 19, 20, 21, 22). Leptin has also been implicated in the regulation of reproductive function as evidenced by: 1) the ob/ob mouse is infertile (23); 2) exogenous leptin advances the first estrus in weanling rodents (24); 3) icv and ip administration of leptin leads to either an increase in LH secretion (25) or prevention of reduced pulsatile LH secretion during fasting (26); and 4) administration of leptin to ob/ob mice restores LH secretion and increases uterine weight and follicular development (15). Furthermore, a recent report has demonstrated that administration of leptin to food deprived lactating rats reduces the period of infertility (27). Thus, leptin appears to be involved in regulation of food intake and is positively correlated with increased LH secretion.
The present study addressed the following questions: 1) Are leptin levels altered during a state of high energy demand such as lactation? 2) Are changes in leptin levels during lactation related to the suckling stimulus and/or the increased energy demand due to milk production? 3) Are changes in leptin levels correlated with the suppression of LH secretion observed during lactation?
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Materials and Methods |
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Exp I: leptin concentrations during chronic lactation
The purpose of this experiment was to determine whether chronic
lactation altered serum leptin concentrations. Group 1 (n = 14)
consisted of adult cycling rats used at the time of diestrus
(Diestrus). Group 2 (n = 18) contained ovarian intact lactating
animals whose litters were adjusted to 8 pups on day 2 post partum
(Lac, Intact). Animals in group 3 (n = 7) were OVX on day 2 post
partum and litters were adjusted to 8 pups (Lac, OVX). Animals in
groups 2 and 3 suckled their pups continuously until collection of
trunk blood on days 9–11 post partum. Measurements of food and water
intake, pup litter body weights, and serum concentrations of leptin,
insulin, PRL and oxytocin were made.
Exp II: effect of the suckling stimulus and/or milk production on
leptin concentrations
The purpose of this set of studies was to determine whether the
suckling stimulus could directly alter leptin levels or whether the
energy drain due to milk production was a factor. To perform these
studies, an acute suckling paradigm was used that has been validated in
our laboratory (12). In this paradigm, animals were OVX on day 2 post
partum and litters were adjusted to 8 pups. The animals were allowed to
continuously suckle their pups until day 9. The litters were removed
for 48 h, beginning on day 9 post partum, to allow the
experimental mothers to return to the nonlactating condition. During
the 48 h period of pup separation, mothers and pups were located
in different parts of the animal facility to avoid the confounding
influences that sight, sound or smell of the pups have on the
experimental mothers. On day 11, the 8-pup litters were returned and
allowed to suckle for 24 h. Ovariectomized animals were used for
these studies because of the ability to measure pulsatile LH secretion
after 48 h of pup separation (see Exp III for a description of the
LH studies).
Control animals (0 pups)
On day 11, after 48 h of pup removal, the control animals
(n = 10) had 0 pups returned.
Acute suckling stimulus, no milk production (Suckling, No
Milk)
On day 9, the pups from these animals (n = 10) were
separated from the mothers and placed onto heating pads in a room
different from the one housing the mothers. Therefore, the pups were
nonfostered during the 48 h period of separation. On day 11, the
experimental mothers had 8 pups returned and were allowed to suckle for
24 h. After the pups were returned to the mothers, and allowed to
suckle for 24 h, there was no evidence of the presence of milk in
the pups’ stomachs (see Results for details).
Acute suckling stimulus, milk production (Suckling, + Milk)
On day 9, the pups from these animals (n = 6) were
separated from the mothers and placed with foster mothers in a
different room. Thus, the pups were fostered during the 48 h
period of separation. On day 11, the experimental mothers had 8 pups
returned and were allowed to suckle for 24 h. At the end of the
24 h suckling period, there was milk present in the pups’
stomachs (see Results for details).
Measurements of food and water intake and pup litter body weights were made on day 9 at the time of pup removal, on day 11 before returning the pups to the experimental mothers, and at the end of 24 h of suckling. At that time, animals were killed and trunk blood was collected and analyzed for serum leptin, insulin, PRL, and oxytocin.
Exp III: changes in leptin and pulsatile LH secretion in response
to acute suckling
The purpose of this experiment was to determine if there was a
link between serum leptin levels and the suppression of LH in response
to acute suckling. On day 2 post partum, animals were OVX and litters
were adjusted to 8 pups. On day 9 post partum, pups were removed from
mothers and were either nonfostered (kept on a heating pad), or
fostered (nursed with foster mothers). Meanwhile experimental mothers
underwent surgery for implantation of chronic right jugular indwelling
catheters as previously described (12). It should be noted that 48
h of pup removal is sufficient to restore pulsatile LH secretion in OVX
lactators. Forty-eight hours after pup removal, the animals were
divided into three groups as follows: Group 1 had no pup replacement (0
Pups, n = 8). Group 2 had 8 pups replaced; the pups had been
nonfostered and kept on heating pads (suckling, No Milk, n = 6).
Group 3 had 8 pups replaced; the pups had spent the 48 h with
foster mothers (Suckling, + Milk, n = 8). After 24 h of
suckling, blood samples were collected from all groups at 6-min
intervals for 3 h. At the end of the sampling period, animals were
killed and brains were frozen for future studies.
Hormone assays
Serum leptin levels were assessed using a rodent specific
sandwich ELISA with a mouse recombinant leptin standard, as previously
described (28). Serum leptin levels are expressed as ng/ml. Insulin
levels were determined using a kit (Linco, St. Louis, MO); the data are
expressed as ng/ml. PRL and oxytocin concentrations were generously
performed by Dr. M. Freeman (Florida State University) and Dr. J.
Verbalis (Georgetown University), respectively, as previously described
(29, 30). Serum PRL is expressed as ng/ml while oxytocin levels are
reported as pg/ml. LH concentrations were determined by RIA using
reagents provided by the NIH, except for the LH antibody, which was
ovine CSU 120 (provided by Dr. G. Niswender at Colorado State
University). The reference standard was NIH RP-3. For the assay, plasma
volumes of 20 µl were used. The sensitivity of the assay was 0.5
ng/ml at a serum volume of 20 µl and between assay variation was
4.9%.
Statistical analysis
Pulsatile LH secretion was evaluated using a personal computer
version of the computer assisted algorithm for the study of episodic
hormone secretion (Pulsar; 31). Data from the Pulsar analysis and
hormone measurements were subjected to either one-way ANOVA with
Newman-Keuls multiple comparison test or Student’s t
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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illustrates leptin levels
in the various animal groups used in this study. When compared with
normal cycling rats during diestrus, chronic lactating rats had a
significant decrease in serum leptin levels. This decrease was not
dependent on the presence of the ovaries as similar results were
obtained in ovarian intact and OVX chronic lactating animals. Insulin
levels were not different when either intact or OVX chronically
lactating animals were compared with diestrous animals. Chronic
lactating rats displayed typical increases in serum PRL and oxytocin
levels, compared with diestrous rats (Fig. 2). Food and water intake did not differ
between intact or OVX lactators and had increased by about 300% and
250%, respectively, by day 9 of lactation, compared with cycling
animals (Table 1).
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illustrates that nonfostered
pups lost weight during the 48 h period of removal from their
mothers (100% weight on day 9 vs. 84% weight on day 11).
Nonfostered pups replaced with their original mothers for 24 h
continued to lose weight, suggesting that the mothers were not
producing milk. This was confirmed by examining the contents of the
pups’ stomachs after the 24 h resuckling period, which showed no
milk present (Table 2). Furthermore, animals that were not producing
milk showed no evidence of milk in their mammary glands and had no
significant increase in either food or water intake during the 24
h suckling period (Table 1
). In contrast, fostered pups replaced with
their original mothers for 24 h had stomachs that were full of
milk and did not lose weight like the nonfostered pups (Table 2, 112%
vs. 79% of day 9 weight, respectively). Also, animals that
did produce milk had evidence of milk in their mammary glands and a
significant increase in food intake compared with the end of the 48-h
removal period. Although there was some weight loss by the fostered
pups after 24 h of suckling, this can be explained by the
observation that it takes 12 h of suckling until milk production
is reinitiated in the experimental mothers. If the pups were allowed to
suckle for 48 h, nonfostered pups continued to lose weight,
whereas fostered pups were back to their original weight, signifying
that the mothers were indeed producing milk (data not shown).
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illustrates that removal of the suckling stimulus for 48
h restored serum leptin levels to the values observed in diestrous
animals. Re-imposition of a suckling stimulus for 24 h that did
not result in milk production led to no change in leptin levels, when
compared with nonsuckled control animals. However, 24 h of
suckling that did produce milk resulted in a marked decrease in serum
leptin levels, similar to those seen during chronic lactation.
Removal of the suckling stimulus for 48 h resulted in a
significant increase in insulin levels when compared with the basal
levels observed during diestrus or chronic lactation (Fig. 1). When the
suckling stimulus was reinitiated without the subsequent production of
milk, insulin levels remained elevated. Reinitiation of suckling that
did produce milk reduced serum insulin to levels seen during chronic
lactation (Fig. 1).
Removal of the suckling stimulus for 48 h decreased serum PRL to
levels significantly different from those observed during chronic
lactation, although PRL did not completely return to baseline levels
(Fig. 2). Reimposition of the suckling stimulus by nonfostered
(Suckling, No Milk) and fostered (Suckling + Milk) pups for 24 h
resulted in an increase in PRL secretion compared with nonsuckled
controls and diestrous animals. However, mean PRL levels in animals
suckled for 24 h and producing milk were significantly lower than
those in the chronic lactating rat (Fig. 2).
Surprisingly, removal of the suckling stimulus for 48 h did not
result in a measurable decrease in oxytocin concentrations (Fig. 2).
Furthermore, oxytocin levels remained elevated in animals suckled for
24 h, whether milk was present or not.
Exp III: changes in leptin and pulsatile LH secretion in response
to acute suckling
Figure 3 shows representative
examples of patterns of pulsatile LH secretion after 24 h of no
suckling (0 pups), of suckling with nonfostered pups (Suckling, No
Milk), or of suckling with fostered pups (Suckling, + Milk). Animals
not receiving the suckling stimulus showed typical patterns of
pulsatile LH secretion (0 pups, Fig. 3, Table 3). Animals suckled for 24 h whether
milk was present or not, showed decreased pulsatile LH secretion
compared with the nonsuckled control animals (Fig. 3). Pulsar analysis
revealed that animals suckled for 24 h had significant decreases
in mean LH concentrations, the duration of LH pulses and the average
baseline levels, regardless of whether milk was produced or not (Table 3). The frequency of LH pulses was significantly reduced in animals
receiving the suckling stimulus but producing no milk. However, the
change in animals receiving the suckling stimulus that produced milk
did not reach statistical significance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results presented in Fig. 1 show that during states of chronic
lactation, the levels of serum leptin are significantly decreased
compared with those of cycling animals. Low levels of leptin may play a
role in the increase in food intake observed during lactation and in
the suppression of pulsatile LH (12, 13), suggesting a correlation
between serum leptin levels and GnRH/LH secretion (32). Furthermore,
our laboratory and others have demonstrated that there is a significant
increase in the amount of NPY synthesis and content within distinct
areas of the hypothalamus (8, 10, 11) during lactation. Also, chronic
increases in NPY have been shown to suppress GnRH/LH secretion
(33, 34, 35). Leptin has also been shown to act at the level of the
hypothalamus to decrease the synthesis and secretion of neuropeptide Y
(NPY; 36, 37). Studies have also demonstrated that leptin can activate
regions of the brain important in energy balance (38) and have
localized leptin receptors to the arcuate nucleus, paraventricular
nucleus, and dorsomedial hypothalamic nucleus, areas implicated in the
regulation of food intake, as well as in the regulation of GnRH
function (39, 40). These results suggest that the decrease in leptin
may play an important role in increasing NPY activity and in inhibiting
GnRH/LH secretion.
The acute suckling paradigm was designed to determine whether the suckling stimulus alone could inhibit leptin, or whether the change in energy balance associated with milk production was a necessary requirement. The results of these studies suggest that the suckling stimulus alone is incapable of affecting changes in leptin levels. It was only when an energy drain occurred due to milk production that leptin was significantly suppressed. It is unknown at this time what signals associated with the initiation of milk production are responsible for the suppression of leptin secretion from adipocytes. The low levels of leptin may be important in allowing the mother to adapt to the lactating condition, by removing a signal for satiety which facilitates the increase in food intake.
The difference in insulin levels between mothers producing milk or not producing milk provides strong support for the idea that the mothers suckling fostered pups are indeed undergoing changes in metabolism due to milk production. The dramatic increase in insulin levels observed in animals whose pups had been removed for 48 h was unexpected. This "overshoot" in insulin levels may in part be due to a change in the metabolic "set point" of the lactating animals. The mothers had been exposed to a high metabolic demand for the 9 day chronic lactation period and had increased food and water intake and fat deposition. To acutely remove the drain of milk production would cause dramatic shifts in the mothers’ energy metabolism. The elevated levels of insulin and leptin would contribute to a restoration of normal levels of food and water intake and fat deposition. The idea that energy utilization is regulated by an internal set point has recently been described in a number of animal and human models of obesity (41, 42, 43). When mothers had their pups removed for 48 h and were allowed to resuckle nonfostered pups for 24 or 48 h, (effectively being without a metabolic drain for 3–4 days), insulin levels were still significantly increased compared with control animals (data not shown).
The leptin and insulin data support the notion that animals suckling fostered pups were undergoing an energy drain due to milk production, in contrast to those animals suckling nonfostered pups. After 24 h of suckling with fostered pups, not only was maternal food intake and pups weight increased over that shown in animals nursing nonfostered pups, but the fostered pups’ stomachs contained large amounts of a milk-like substance. In another set of experiments, it was determined that this milk had to be coming from the experimental mothers and was not residual milk from the fostering experience. Once removed from their mothers, it takes approximately 15 h for the pups’ stomachs to become completely void of milk (data not presented). Finally, there was no visual evidence of milk in the mammary tissue of the suckling + no milk group, while in the suckling + milk group, a milky substance was present (data not shown). Therefore, the presence of milk in both the mammary tissue and the pups’ stomachs after 24 h of suckling fostered pups indicates that the experimental mothers had reinitiated milk production and, therefore, were experiencing an energy drain.
At the present time, it is unknown why the mothers suckling nonfostered
pups did not produce milk. PRL and oxytocin levels were elevated,
suggesting that the neural impulses from suckling did act at the level
of the hypothalamus. It is also possible that sensory stimuli from the
pups may have contributed significantly to the increase in PRL and
oxytocin. It is important to mention that the levels of PRL are
decreased significantly in OVX lactators compared with intact lactators
(Fig. 2). This is due in part to the effects of ovarian steroids and
specifically progesterone (44, 45). The opposite is true for oxytocin
levels when intact and OVX lactators are compared. Although the levels
of oxytocin are not significantly different, there was a trend for
oxytocin concentrations to be higher in OVX lactators, again due to
withdrawl of ovarian steroids (46, 47, 48). Clearly, the levels of these
hormones are not good predictors of milk production by the mothers. It
is possible that the intensity of the suckling stimulus may play an
important role. The nonfostered pups were in a weakened state; thus the
intensity of the suckling stimulus, although of sufficient strength to
elicit PRL and oxytocin secretion and to inhibit LH secretion, may not
have been of sufficient strength and duration to elicit milk
production.
The acute suckling model also permitted us to examine the relationship
between leptin levels and the inhibition of LH secretion in response to
suckling. The results demonstrate that a decrease in leptin is not a
necessary prerequisite for the suppression of LH secretion. In response
to an acute suckling stimulus, LH secretion was significantly decreased
after 24 h (Fig. 3), regardless of whether leptin levels decreased
or remained at levels equal to those observed in nonsuckled animals or
cycling animals (Fig. 1). Therefore, this data suggests that the
suckling stimulus alone is capable of suppressing LH secretion without
any alteration in leptin levels or metabolic status. In contrast, the
suckling stimulus alone does not alter leptin secretion. Instead, the
decrease in leptin secretion appears to be occurring in response to the
change in energy balance of the animal. However, since it is currently
not possible to precisely measure the suckling intensity of the two
groups of pups, we cannot rule out that differences in intensity of the
suckling stimulus may have made some contribution.
Overall, these studies demonstrate that during chronic lactation, serum leptin levels are decreased; this decrease appears to be related to the energy drain of milk production and not to the suckling stimulus itself. The decrease in leptin is correlated with the suppression of serum LH levels. However, during an acute suckling stimulus, the effects of suckling are able to suppress serum LH levels in the absence of any decrease in serum leptin levels. These data do not rule out the possibility that in the chronic lactating rat, leptin is one of several factors that contribute to the continued suppression of LH secretion. We are addressing this issue in ongoing studies in which leptin is administered to the chronic lactating rat and LH secretion is measured. We conclude that, although leptin may contribute to the long-term suppression of GnRH/LH secretion during lactation, it is not a requisite component in the response to the suckling stimulus.
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Acknowledgments |
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Footnotes |
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Received October 19, 1998.
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