1 Max-Planck-Institut für
physiologische und klinische Forschung, To determine the degree to which the leptin
receptor mutation (fa) influences
the responsiveness to leptin during the first postnatal week, we
injected recombinant leptin (600 pmol · g
Zucker rats; genetic obesity; heterozygous difference; development; juvenile rats
THE ROLE OF LEPTIN found in the plasma of newborn and
suckling-age rats (5) is obscure for several reasons. One reason is
that fat storage in white and brown adipose tissue starts only after
the first suckling (16, 19). Other reasons are the immaturity of the
brain of the newborn rat and the peculiarities of ingestive control in
suckling-age pups (12, 21), which together would seem to question the
suggested role of leptin in the control of food intake during the first
days of life (5). Still another reason is a sudden pronounced increase
of plasma leptin levels that occurs, without a developmental increase
in body fat content, in 7- to 12-day-old mice (1, 6) and, to a much
smaller extent, in 10-day-old rats (18). These observations prompted
the suggestion that leptin in newborn rodents serves functions
different from its later role in the regulation of body fat stores (1):
specifically, that it plays a role in the development and function of
the neuroendocrine axis (1).
Unexpectedly in view of the above findings, however, we observed a
close correlation between plasma leptin levels and total fat mass of
individual rat pups at 10 days of age (31). Moreover, treating rat pups
with leptin from 7 to 16 days of age decreased their body fat content
by an amount closely correlated with the logarithm of the leptin dose
(24), not by decreasing food intake but by increasing thermoregulatory
energy expenditure (25, 26). Because both the milk intake and the
average daily metabolic rate of rat pups during the first postnatal
week are hard to evaluate precisely enough, we instead investigated the
effect of recombinant leptin on total body fat content, a quantity that
not only can be evaluated very precisely (13) but that also reflects
the variable outcome of energy exchange more directly.
Evaluation of the leptin responsiveness of rats throughout the first
postnatal week is also interesting because pups homozygous for the
leptin receptor defect (fa; Ref. 3)
already deposit excessive amounts of fat during the first postnatal
week (14, 22, 29). Moreover, at the end of the first postnatal week, the body fat content difference between wild-type (+/+) and
heterozygous (+/fa) rats is nearly
one-half as big as that between wild-type and fatty
(fa/fa) pups (22, 29). Thus the
fa mutation, which in adult rats
appears to be recessive, seems in young rats to be expressed in a
codominant way (29). These observations might indicate that in the very
first days of life leptin already plays a role in a feedback system
controlling body fat. It has, however, also to be considered that the
leptin receptor itself, particularly in the early developmental stages,
might serve functions beyond regulating the body fat stores by
transducing the circulating leptin levels (1, 31). If so, the
fa mutation might cause early
abnormalities in the regulation of body fat content that are not due to
the feedback loop, i.e., fat cell size, leptin concentration, and
hypothalamic leptin receptors, that is disturbed later in life. To find
out whether or not a disturbed leptin responsiveness already plays a
role in the earliest stages of excessive fat deposition, we compared
the effects of recombinant leptin, injected throughout the first
postnatal week in doses at the upper end of the physiological range
(24), on the growth and body fat content of rat pups carrying 0, 1, or
2 copies of the fa allele.
Animals
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 · day
1
sc from day
1 to
day
7) into wild-type (+/+),
heterozygous (+/fa), and fatty
(fa/fa) rat pups. Growth and final
body fat content of these leptin-treated pups were compared with those
of saline-treated littermates of the same genotype. The body mass of
the leptin-treated +/+ pups, but not that of the
+/fa and
fa/fa pups, increased more slowly than
that of their respective controls, and fat content at
day 7 was reduced by 37% in +/+ pups, by 22% in
+/fa pups, but not at all in
fa/fa pups. Plasma leptin remained
excessively high throughout the day under this treatment, but a 30-fold
lower leptin dose, causing only moderate changes of plasma leptin,
still reduced the body fat of +/+ pups significantly. We conclude that leptin participates in the control of even the earliest stages of fat
deposition and that the response to supraphysiological doses of leptin
is markedly reduced in 1-wk-old pups with one fa allele and absent in pups with two
fa alleles.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Genotype Identification
Immediately after the first suckling, a small tissue sample was removed by a tail cut. Rat genomic DNA was isolated after the protocol of a commercially available kit (QIAmp Tissue Kit, Qiagen, Hilden, Germany). Genotypes at the leptin-receptor locus were then identified as described by Chua et al. (3). In short, a PCR was conducted in a reaction volume of 50 µl containing 200 µM deoxynucleoside 5'-triphosphates (Boehringer Mannheim, Mannheim, Germany), 20 pmol of each primer (MWG, Ebersberg, Germany), 2.5 U Taq polymerase (Boehringer), 10× PCR buffer containing 15 mM MgCl2 (Boehringer), and 2 µl rat genomic DNA. Amplifications in a thermocycler (Gene Amp PCR System 2400, Perkin Elmer, Weiterstadt, Germany) consisted of an initial denaturation at 94°C for 5 min; 35 cycles of 94°C, 55°C, and 72°C, each for 30 s; and a final extension of 72°C for 7 min. Then 15 µl of the resulting PCR product were digested by 5 U Msp I (Boehringer) for 4 h at 37°C. Digestion products were resolved by nondenaturating polyacrylamide gel electrophoresis and ethidium bromide staining.Experimental Protocol and Body Composition Analysis
We used recombinant His-tagged murine leptin (17,600 Da) produced as described previously (26). Starting when the animals were 1 day old (that is, on the 2nd postnatal day), one-half of the littermates of any given genotype received subcutaneous injections of leptin (600 pmol · gPlasma Leptin Determinations and Control Experiments
Plasma leptin concentrations were determined in some of the wild-type litters at the end of the treatment period and in several more litters under different physiological conditions. To test whether or not physiological changes result in measurable changes of leptin levels at this age, we isolated two wild-type litters of 7-day-old pups for 6-9 h from their mothers. The pups were allowed to huddle in the nest, and whereas in one-half of them (n = 7) a normal stomach filling was maintained by feeding them one or two times with an artificial milk, the others (n = 8) received only water until their stomachs were totally empty. Additionally, one-half of the pups of another litter were treated with a low dose of leptin (20 pmol · gEvaluation
The SigmaStat program was used to perform two-way ANOVA, if necessary, of the repeated measurement design. Average data values were described accordingly by least square means ± SE provided by ANOVA.Growth. The statistical significance of differences in the growth between leptin-treated and control pups of each genotype was evaluated by two-way ANOVA for repeated measurements (with age and treatment as the factors). This could be done because in none of our previous studies had any indication for a litter × genotype interaction (or a litter-gender × genotype interaction, see next paragraph) in growth or body composition been detected (13, 14, 22, 26).
Presentation of absolute values. To present final body composition, carcass mass, and body mass at each age, we took into account the considerable between-litter variability in growth (13, 14, 22, 26, 29) as well as the small gender-related differences in these variables (29), which might already be evident in 7-day-old pups. This was done in a way similar to that used in an earlier study (22): for each genotype, we separated the treatment effect from possible gender and litter effects by considering a combined litter-gender effect as the second factor in the two-way ANOVA. This can be easily done by assigning different litter numbers to male and female littermates. In this way, we could thus clearly separate the treatment effects from the highly significant litter-gender effect. We did not, however, try to separate the gender and litter effects because in litters containing 8-12 pups with a more or less uneven gender distribution, this separation would have required the analysis of data from a huge number of litters and the outcome of such an analysis would not have been important for the aim of this study.
Percent changes in body composition. In addition to evaluating the absolute amounts of fat, FFDM, and water, we also evaluated the changes in these quantities by expressing the data of each pup as a percentage of the mean values of its control littermates of the same genotype.
Evaluation by regression analysis. To evaluate the statistical significance of leptin-induced changes in body composition between +/+ and +/fa pups reared in different litters, we used regression analysis. Regression analysis was also applied to demonstrate for the different genotypes the close correlation between body mass and body fat content irrespective of interlitter variability.
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RESULTS |
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Leptin Effects in Wild-Type and Heterozygous Pups
The body mass of the leptin-treated +/+ pups increased more slowly than that of their control littermates (P < 0.01 for the interaction of age and treatment in two-way ANOVA for repeated measurements) starting from identical body masses. With regard to age, the difference between the treated and control pups first became statistically significant when the pups were 3 days old as illustrated by Fig. 1A. The body mass of the +/fa pups, in contrast, was not affected by the leptin treatment (P = 0.7 for the interaction of age and treatment in two-way ANOVA for repeated measurements), and separate analysis of data for each age group confirmed that body mass of the leptin-treated +/fa pups never fell significantly below that of their control littermates (Fig. 1B).
|
Leptin markedly affected the final body fat content of the +/+ pups,
which was 0.32 g less (Table
1) or nearly 37% lower (Fig.
2) than that of their control littermates.
The FFDM of these leptin-treated pups was only (but still
significantly) 0.09 g (3%) below that of the controls, and this
decrease in FFDM was accompanied by a significant decrease in body
water (Table 1, Fig. 2). These changes in both body fat and lean body
mass thus accounted for the significant changes in final body mass and
carcass mass (Table 1, Fig. 1A).
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|
Leptin-treated heterozygous pups also had a total body fat content significantly less than that of the heterozygous controls (Table 1), but the difference between these treated and control pups was only about one-half of that between the treated and control wild-type pups (Fig. 2). Moreover, FFDM, body water content, final body mass, and carcass mass (Table 1, Fig. 1B) of the leptin-treated heterozygous pups were not different from those of their control littermates (all P > 0.05).
To take into account the large interlitter differences in body fat
content when evaluating the effect of leptin on wild-type and
heterozygous pups that were not reared in the same litters, we plotted,
for each litter, the average body fat content of leptin-treated wild-type and heterozygous pups against the average body fat content of
the corresponding control littermates (Fig.
3A). For
both genotypes, the final body fat content of the treated pups was
closely correlated with that of their control littermates and was
clearly shifted below the line of identity. The regression line for the
+/+ pups, however, is significantly below that of the
+/fa pups
(P < 0.05 for the
y-intercept of parallel lines). In
addition, because the average body fat content of the pups in some of
the litters containing only +/fa pups
was rather low, it is important to note that the size of the effect of
leptin is not a function of the average fat content of the control
animals in the litter (Fig. 3B).
Moreover, independent of the interlitter variability of body fat
content, in both genotypes, the absolute amount of body fat and body
mass in all leptin-treated pups on the one hand and all control pups on
the other hand was closely correlated (Fig.
4, A and
B).
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|
No Leptin Effect in fa/fa Pups
The body mass of the leptin-treated fa/fa pups did not differ from that of their controls throughout the entire treatment period (Fig. 1C), and neither the body fat content nor the FFDM of the treated fa/fa pups was less than that of their control fa/fa littermates (Table 1, Fig. 2). To confirm that the complete absence of a leptin effect in fa/fa pups was not due to some uncontrolled difference between litters, we plotted, separately for each genotype, the body fat content of all leptin-treated and control pups against their body mass (Fig. 4). The regression lines of leptin-treated and control fa/fa pups were identical, whereas the regression lines for leptin-treated +/+ and +/fa pups were significantly shifted below the lines for the respective controls. Moreover, the regression lines for the correlation between body fat and body mass found for the fa/fa and +/fa control pups from this study did not differ significantly from those found 3 years ago for pups of the same genotype (14).Control Experiments: Time Course of Plasma Leptin Levels and Responsiveness of +/+ Pups to Lower Leptin Dose
Plasma leptin concentration of untreated and saline-treated +/+ control pups ranged between 1 and 6 ng/ml (2.7 ± 0.2; n = 25). Plasma leptin levels were similar among littermates but differed considerably between litters without correlation to body mass or body fat content. Within the same litter, leptin levels of postresorptive pups (1.3 ± 0.2 ng/ml) were significantly lower than those of milk-fed littermates (2.4 ± 0.2 ng/ml; P < 0.002, 2-way ANOVA). Plasma leptin levels after treatment with 600 pmol · g ![]() |
DISCUSSION |
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Leptin Responsiveness During the First Postnatal Weeks
The continuous separation of the body mass of leptin-treated +/+ pups from that of their control littermates throughout the entire treatment period (Fig. 1A) shows that leptin is already effective in the very first postnatal days and that the leptin responsiveness does not change dramatically during the first postnatal week. At the end of the 6-day treatment period, the body fat content of leptin-treated +/+ pups was 37% less than that of their control littermates. This reflects even a slightly larger per-day decrease in body fat content than the 44% decrease found after a 9-day treatment period in rat pups that had been treated with the same dose of leptin from 7 to 16 days (26) and demonstrates that the leptin responsiveness during the first postnatal week is higher, rather than lower, than that during the second postnatal week. This notion is supported by the control experiments with +/+ pups given a 30-fold lower dose: in this case the body fat content of 16-day-old pups, after being treated as previously mentioned, was 15% lower than that of their controls (24), whereas we observed a decrease of 26% after the treatment from day 1 through day 7.Physiological Relevance of the Response
With 600 pmol · gHeterozygous Difference
Previous studies have shown that both the body mass and fat content of 7-day-old heterozygous pups are markedly higher than those of wild-type littermates and that there is an extremely strong litter effect on these variables (22, 29). Most of the experiments in this study, however, used pups in litters containing only one genotype to facilitate the within-litter comparison of leptin-treated pups and controls, and a direct comparison of body composition between our +/+ and +/fa pups would therefore not be meaningful. Regression analysis, however, allowed us to compare the effects of leptin between wild-type and heterozygous pups while taking into consideration the variation in the average fat content of the pups in each litter (see Fig. 3). This analysis confirmed that the leptin effect in the heterozygous pups is smaller than that in the wild-type pups and that this smaller effect is not an artifact due to differences in the average litter fat content of wild-type and heterozygous pups reared in different litters. Previous studies in 2-wk-old pups, in contrast, had not found a heterozygous difference in responsiveness to leptin (Ref. 26; O. Stehling and I. Schmidt, unpublished observations), but this is not surprising because the heterozygous difference in spontaneous fat deposition does not increase between 7 and 16 days of age (22, 29).Absence of Leptin Responsiveness in Suckling-Age fa/fa Pups
At least during the first two postnatal weeks, the homozygous occurrence of the fa mutation (3) seems to completely eliminate the physiological function of the leptin receptor in the control of body fat stores (present results and Ref. 24). This is most clearly demonstrated by the identity of the regression lines of body fat on body mass of leptin-treated and control fa/fa pups at 16 days of age (24) as well as at 7 days of age (Fig. 4C). At the same time, the normal growth rate and unchanged fat content of our leptin-treated fa/fa pups represent a control so that the protein injection per se did not have adverse effects on pups of this age.Adult fa/fa rats have been reported to
have a decreased leptin responsiveness but to still show some responses
to high doses injected into the cerebrospinal fluid (4, 23). Neither
2-wk-old pups (26) nor the pups in the present study, however, showed any decrease in body fat content when treated with 600 pmol · g1 · day
1
of leptin injected subcutaneously. It might thus be argued that we
might have seen some response in our pups if we had used higher doses,
because the effective subcutaneous doses of leptin required in adults
are reported to be ~250- to 1,000-fold greater than the effective
intracerebroventricular doses (9, 27). The high dose used by us is,
however, ~1,000 times the lowest intracerebroventricular dose
effective in adult fa/fa rats (4) and
is 30 times the subcutaneous dose that still produced a marked decrease
(26 and 15%, respectively) in the body fat content of 1- and 2-wk-old wild-type rats (present study and Ref. 26). It thus seems unlikely that
the observed complete absence of any leptin-induced change in body fat
content of 1- and 2-wk-old fa/fa pups
is due to an insufficient dosage.
Physiological Relevance of Decreased Leptin Responsiveness During the First Postnatal Week
The severity of the fa lesion during the first week of life is emphasized not only by the lack of a response of the fa/fa pups to leptin, but also by the markedly reduced responsiveness of the +/fa pups compared with that of the wild-type pups. The fa lesion appears to be a largely recessive trait in adult animals, but even one defective allele halves the effect of leptin in 1-wk-old rat pups. Leptin-receptor-mediated transport routes might not be fully functional in animals carrying the fa lesion (30), and the clear effect of the leptin receptor mutation throughout the first postnatal week is therefore particularly interesting because the leaky blood-brain barrier of the newborn rat (10) can hardly be expected to provide a perfect barrier to circulating leptin. Another advantage of using newborn rats when investigating relations between leptin responsiveness and the fa mutation is that secondary differences like changes in fat cell size and plasma insulin levels that might be expected to influence the responsiveness of an older fa/fa rat to leptin (28) can be excluded. The present results show that the consequences of the fa mutation in the leptin receptor are especially severe in the first postnatal week even though the total amount of fat stored and, consequently, the size of genotype differences in fat storage are still very small.Early Detection of Differences in Body Fat Content
Because the identification of leptin and its receptor mutations has triggered a surge of investigations in early life, it seems important to clarify here the methods that have allowed us to reliably assess small differences in body fat content as well as in other parameters reflecting the energy balance of suckling-age rat pups. We have used whole body chloroform extraction to measure body fat because it is a simple and reliable method (13, 14) and is not susceptible to the mixing and sampling errors that can occur when only aliquots are analyzed. Moreover, when mean values are calculated, statistical consideration of litter effects (17, 22, 29) in simultaneously measured variables rules out a lot of possible methodological variations and also neutralizes many other possible variations that might confound the results: genetic background, litter size, and age of the mother. Thus small genotype differences in energy balance can be filtered out of the confounding variability. As far as body composition is concerned, another way to overcome many of these problems is regression analysis. This is because the genotype differences are very consistent across litters and even across studies conducted years apart (14), and despite large interlitter variability in growth and body fat content, no litter × genotype interactions occur (13, 14, 22).Perspectives
Ahima et al. (1) have put forward the interesting hypothesis that the sudden dramatic increase in plasma leptin concentrations seen in 1-wk-old mice, and to some extent also in rat pups (18), triggers the cascade of hormonal changes that prepare the pups for weaning. At the same time the lack of a correlation between the leptin surge and a developmental increase in percent body fat, as well as the absence of decreases in the leptin levels of pups food deprived for 12 h under undefined thermal conditions, has been thought to indicate that leptin is not involved in a feedback control of body fat stores in early developmental stages (1). We have found, on the other hand, clear plasma leptin differences in postresorptive and fed pups huddling within the same litter. More importantly, however, we should remember that the way the size of fat stores is transduced into a "leptin signal" is still a mystery and the acute changes of leptin levels that in adult animals are associated with feeding cycles and ambient temperature changes (28) are a complication rather than a necessity for a feedback signal thought to reflect total body fat content. Moreover, how the close correlation between body fat content and plasma leptin levels that is evident in population data can be explained in terms of the different rates of leptin production in different tissues and the acute changes of leptin levels that are associated with temporary changes in the rate of fat deposition (15, 18, 28) is still totally unclear. It has to be expected that the sensitivity of the receptors is adjusted if leptin is to provide a feedback signal despite the changes occurring during the juvenile leptin surge. Adaptive changes of receptor sensitivity associated with changing hormone levels are, however, well known. An important developmental function of leptin like that suggested by Ahima et al. would thus not exclude the role of leptin in the early postnatal control of body fat content indicated by the present study. More detailed studies are therefore needed on the normal changes of leptin concentrations of sucklings in response to physiological stimuli that take into account the particularities in the thermoregulation (11, 13, 21, 26) and feeding behavior (12) of the sucklings. ![]() |
ACKNOWLEDGEMENTS |
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We are grateful to Randy Kaul for continuing service as our native English-speaking person and to Johann Ertl (Hoechst Marion Roussel, Frankfurt, Germany) for careful production of the leptin. We also thank many people for helping at various stages of the experiments, particularly Oliver Stehling, Martin Olbort, Roswitha Bender, and Heiko Döring.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (Schm 680/2-3).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. Schmidt,
Max-Planck-Institut für physiologische und
klinische Forschung, W. G. Kerckhoff-Institut, Parkstrae
1, D-61231 Bad Nauheim, Germany (E-mail:
I.Schmidt{at}kerckhoff.mpg.de).
Received 31 August 1998; accepted in final form 14 January 1999.
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