Leptin responsiveness and gene dosage for leptin receptor mutation (fa) in newborn rats

Susanne Kraeft1, Knut Schwarzer1, Sandra Eiden1, Barbara Nuesslein-Hildesheim1, Gerald Preibisch2, and Ingrid Schmidt1

1 Max-Planck-Institut für physiologische und klinische Forschung, W. G. Kerckhoff-Institut, D-61231 Bad Nauheim; and 2 Hoechst Marion Roussel, DG Metabolism Frankfurt, D-65926 Frankfurt, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-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.

Zucker rats; genetic obesity; heterozygous difference; development; juvenile rats


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

We used Zucker rat (13M) and Zucker rat × Brown Norway hybrid pups from 18 litters reared in our colony (14) maintained at 22°C with lights on from 0600 to 1800. We used as well the offspring of two +/+ parents (4 litters), the offspring of +/+ females and +/fa males (4 litters), the offspring of +/+ females and fa/fa males (4 litters), and the offspring of +/fa females and fa/fa males (6 litters). On the day of birth (day 0) each pup was marked with subcutaneous injections of India ink, and the genotypes of the offspring of a heterozygous parent were determined before the pups were assigned to treatment and control groups. Apart from the brief treatment and weighing procedures, the pups were left with their mothers.

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 · g-1 · day-1) in two divided doses daily at about 0800 and 1800; the other one-half received control injections (phosphate-buffered saline). Within each litter, the pups with the same genotype were randomly assigned to the treatment and control groups in such a way that the average body masses of the leptin-treated animals before the first injection exactly matched that of their control littermates. Whenever the average masses of the two groups did not match exactly (differences usually <0.2 g), the slightly heavier one was treated with leptin to prevent a leptin effect from being indicated artifactually as a result of smaller animals growing more slowly. Pups were weighed each evening and, when they were 7 days old, were weighed in the afternoon before they were killed. Carcass mass was determined after the stomach and intestines were removed, and body composition [fat content and fat-free dry mass (FFDM)] was evaluated by drying to constant weight, followed by whole body chloroform extraction in a Soxhlet apparatus (13).

Plasma 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 · g-1 · day-1) as described above. To determine the time course of plasma leptin changes after the injections, pups from four more wild-type litters received two daily injections of leptin (either 600 or 20 pmol · g-1 · day-1) on postnatal day 6 and were then killed at various times after the last injection. Pups were anesthetized by CO2 and decapitated. Blood was collected on ice in tubes containing heparin as anticoagulant. Dilution was determined by weighing, and concentrations were appropriately corrected. Plasma was collected after centrifugation and stored at -80°C until leptin concentrations were determined with a commercial leptin RIA kit (Linco, St. Charles, MO).

Evaluation

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   Least square means (±SE bars would be smaller than symbols) determined by 2-way ANOVA (with litter-gender and treatment as factors) are used to illustrate differences in body mass of wild-type (+/+, A), heterozygous (+/fa, B), and fatty (fa/fa, C) leptin-treated (, black-triangle, ) and control (open circle , triangle , ) pups separately for each day throughout 1st postnatal week. * P < 0.05 for treatment effect.

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Carcass mass, FFDM, fat mass, and water mass of +/+, +/fa, and fa/fa pups treated with leptin or phosphate-buffered saline



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Average difference (means ± SE) in body fat, fat-free dry mass (FFDM), and water content between leptin-treated pups and control littermates of same genotype. Values are a percentage by which data of leptin-treated pups differ from mean values of fat mass, FFDM, and water mass in control littermates of same genotype. Forward-hatched bars, +/+; back-hatched bars, +/fa; cross-hatched bars, fa/fa.

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).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   For each litter, average fat content of leptin-treated pups (A) and average leptin-induced change in fat content (B) are shown as a function of average fat content of their control littermates. Averages for wild-type () and heterozygous (triangle ) pups were calculated separately. Leptin-induced changes in litter fat content are not significantly correlated with average litter fat content of control pups (r = 0.08 for +/fa and 0.11 for +/+).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Final body fat content of leptin-treated (, black-triangle, ) and control (open circle , triangle , ) pups as a function of carcass mass. A, +/+; B, +/fa; C, fa/fa pups.

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-1 · day-1 were still between 50 and 100 ng/ml 8 h after an injection and ~10 ng/ml 12 h after an injection. In the pups treated with the 30-fold lower dose, however, plasma leptin levels had returned after 6 h to the physiological range (3.6 ± 0.5 ng/ml; n = 4) and were after 8 h (2.5 ± 0.4 ng/ml; n = 4) and 12 h (2.2 ± 0.4 ng/ml; n = 8) no longer significantly different from those of the controls. Treatment of wild-type pups from day 1 to day 7 with the low dose resulted in a 26% lower body fat content (P < 0.05, ANOVA) in comparison with their control littermates (Table 1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · g-1 · day-1, we chose a dose increasing plasma leptin beyond the physiological range, because we wanted to have a good chance of seeing clear genotype differences within only 6 days. Because bolus injections produce large but brief increases in plasma leptin concentration (2), their effects on leptin concentrations in other body fluid compartments where leptin might act (e.g., the cerebrospinal fluid) are unclear, and determination of transient plasma levels is therefore meaningless. However, the plasma leptin measurements indicated that at the high dose plasma levels were, even 8 h after the injection, still more than 10-fold above the physiological level found in +/+ control pups and did not return to normal before the next injection. With the 20 pmol · g-1 · day-1 dose, however, plasma leptin levels were within the physiological range 6 h after the injections and were at or even slightly below the level of control pups ~8-12 h later. Nevertheless, it seems important to consider the plasma levels that result when the same dose is applied continuously via miniosmotic pumps so that steady-state plasma levels can be determined. This is, however, technically possible only in older animals. Doses of ~20 pmol · g-1 · day-1 applied via miniosmotic pumps to lean adult mice (9) or to 15- to 24-day-old rat pups (8) caused plasma leptin levels to increase two- to fivefold. That is, they stayed within the physiological range.

Heterozygous 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 · g-1 · 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

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.


    FOOTNOTES

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, Parkstrabeta e 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahima, R. S., D. Prabakaran, and J. S. Flier. Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding. J. Clin. Invest. 101: 1020-1027, 1997[Abstract/Free Full Text].

2.   Ahima, R. S., D. Prabakaran, C. Mantzoros, D. Qu, B. Lowell, E. Maratos-Flier, and J. S. Flier. Role of leptin in the neuroendocrine response to fasting. Nature 382: 250-252, 1996[Medline].

3.   Chua, S. C., Jr., D. W. White, X. S. Wu Peng, S.-M. Liu, N. Okada, E. E. Kershaw, W. K. Chung, L. Power-Kehoe, M. Chua, L. A. Tartaglia, and R. L. Leibel. Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr). Diabetes 45: 1141-1143, 1996[Abstract].

4.   Cusin, I., F. Rohner-Jeanrenaud, A. Stricker-Krongrad, and B. Jeanrenaud. The weight-reducing effect of an intracerebroventricular bolus injection of leptin in genetically obese fa/fa rats. Diabetes 45: 1446-1450, 1996[Abstract].

5.   Dessolin, S., M. Schalling, O. Champigny, F. Lönnqvist, G. Ailhaud, C. Dani, and D. Ricquier. Leptin gene is expressed in rat brown adipose tissue at birth. FASEB J. 11: 382-387, 1997[Abstract/Free Full Text].

6.   Devaskar, U., C. Ollesch, R. A. Rajakumar, and P. A. Rajakumar. Developmental changes in ob gene expression and circulating leptin peptide concentrations. Biochem. Biophys. Res. Commun. 238: 44-47, 1997[Medline].

7.   Döring, H., K. Schwarzer, B. Nuesslein-Hildesheim, and I. Schmidt. Leptin selectively increases energy expenditure of food-restricted lean mice. Int. J. Obes. 22: 83-88, 1998[Medline].

8.  Eiden, S., M. Olbort, A. Tripp, B. Nuesslein-Hildesheim, and I. Schmidt. Developmental changes of the rat's response to recombinant leptin (Abstract). Int. J. Obes., 22, Suppl. 3: 35, 1998.

9.   Halaas, J. L., C. Boozer, J. Blair-West, N. Fidahusein, D. A. Denton, and J. M. Friedman. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc. Natl. Acad. Sci. USA 94: 8878-8883, 1997[Abstract/Free Full Text].

10.   Johanson, C. E. Implications of the Blood-Brain Barrier and its Manipulation, edited by A. Edward, and M. D. Neuwelt. New York: Plenum, 1989, vol. 1, p. 157-198.

11.   Körtner, G., K. Schildhauer, O. Petrova, and I. Schmidt. Rapid changes in metabolic cold defense and GDP binding to brown adipose tissue mitochondria of rat pups. Am. J. Physiol. 264 (Regulatory Integrative Comp. Physiol. 33): R1017-R1023, 1993[Abstract/Free Full Text].

12.   Lorenz, D. N. Suckling physiology and behavior of rats: an integrated theory of ingestion and satiety. Prog. Psychobiol. Physiol. Psychol. 15: 1-83, 1992.

13.   Markewicz, B., G. Kuhmichel, and I. Schmidt. Onset of excess fat deposition in Zucker rats with and without decreased thermogenesis. Am. J. Physiol. 265 (Endocrinol. Metab. 28): E478-E486, 1993[Abstract/Free Full Text].

14.   Meierfrankenfeld, B., M. Abelenda, H. Jauker, M. Klingenspor, E. E. Kershaw, S. C. Chua, Jr., R. L. Leibel, and I. Schmidt. Perinatal energy stores and excessive fat deposition in genetically obese (fa/fa) rats. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E700-E708, 1996[Abstract/Free Full Text].

15.   Pelleymounter, M. A. Leptin and the physiology of obesity. Curr. Pharmaceut. Des. 3: 85-98, 1997.

16.   Péquignot-Planche, E., P. De Gasquet, A. Boulangé, and N. T. Tonnu. Lipoprotein lipase activity at onset of development of white adipose tissue in newborn rats. Biochem. J. 162: 461-463, 1977[Medline].

17.   Planche, E., M. Joliff, and R. Bazin. Energy expenditure and adipose tissue development in 2- to 8-day-old Zucker rats. Int. J. Obes. 12: 353-360, 1988[Medline].

18.   Rayner, D. V., G. D. Dalgliesh, J. S. Duncan, L. J. Hardie, N. Hoggard, and P. Trayhurn. Postnatal development of the ob gene system: elevated leptin levels in suckling fa/fa rats. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R446-R450, 1997[Abstract/Free Full Text].

19.   Ricquier, D., and P. Hemon. A study of phospholipids and triglycerides in several tissues of the rat during fetal and neonatal development. Biol. Neonate 28: 225-240, 1976.

20.   Schmidt, I., H. Döring, O. Stehling, B. Nuesslein-Hildesheim, S. Steinlechner, and K. Schwarzer. Leptin disinhibits rather than stimulates sympathetically mediated energy expenditure. In: Leptin---The Voice of the Adipose Tissue, edited by W. F. Blum, W. Kiess, and W. Rascher. Heidelberg, Germany: Barth, 1997, p. 133-139.

21.   Schmidt, I., R. Kaul, and G. Heldmaier. Thermoregulation and diurnal rhythms in 1-week-old rat pups. Can. J. Physiol. Pharmacol. 65: 1355-1363, 1987[Medline].

22.   Schwarzer, K., H. Döring, and I. Schmidt. Different physiological traits underlying increased body fat of fatty (fa/fa) and heterozygous (+/fa) rats. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E100-E106, 1997[Abstract/Free Full Text].

23.   Seeley, R. J., G. van Dijk, L. A. Campfield, F. J. Smith, P. Burn, J. A. Nelligan, S. M. Bell, D. G. Baskin, S. C. Woods, and M. W. Schwartz. Intraventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats. Horm. Metab. Res. 28: 664-668, 1996[Medline].

24.   Stehling, O., H. Döring, B. Nuesslein-Hildesheim, M. Olbort, and I. Schmidt. Leptin-doses halving the body fat of lean pups are not effective in genetically obese (fa/fa) rat pups. In: Leptin---The Voice of the Adipose Tissue, edited by W. F. Blum, W. Kiess, and W. Rascher. Heidelberg, Germany: Barth, 1997, p. 140-147.

25.   Stehling, O., H. Döring, B. Nuesslein-Hildesheim, M. Olbort, and I. Schmidt. Leptin does not reduce body fat content but augments cold defense abilities in thermoneutrally reared rat pups. Pflügers Arch. 434: 694-697, 1997[Medline].

26.   Stehling, O., H. Döring, and I. Schmidt. Leptin reduces juvenile fat stores by altering the circadian cycle of energy expenditure. Am. J. Physiol. 271 (Regulatory Integrative Comp. Physiol. 40): R1770-R1774, 1996[Abstract/Free Full Text].

27.   Stephens, T. W., M. Basinski, P. K. Bristow, J. M. Bue-Vallesky, S. G. Burgett, L. Craft, J. Hale, J. Hoffmann, H. M. Hsiung, A. Kriauciunas, W. MacKellar, P. R. Rosteck, Jr., B. Schoner, D. Smith, F. C. Tinsley, X.-Y. Zhang, and M. Heiman. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 377: 530-532, 1995[Medline].

28.   Trayhurn, P., and D. V. Rayner. Hormones and the ob gene product (leptin) in the control of energy balance. Biochem. Soc. Trans. 24: 565-570, 1996[Medline].

29.   Truett, G. E., R. J. Tempelman, and J. A. Walker. Codominant effects of the fatty (fa) gene during early development of obesity. Am. J. Physiol. 268 (Endocrinol. Metab. 31): E15-E20, 1995[Abstract/Free Full Text].

30.   Wu-Peng, X. S., S. C. Chua, Jr., N. Okada, S.-M. Liu, M. Nicolson, and R. Leibel. Phenotype of the obese Koletsky (f) rat due to Tyr763stop mutation in the extracellular domain of the extracellular domain of the leptin receptor (Lepr). Diabetes 46: 513-518, 1997[Abstract].

31.   Zhang, Y., M. Olbort, K. Schwarzer, B. Nuesslein-Hildesheim, M. Nicolson, E. Murphy, T. J. Kowalski, I. Schmidt, and R. Leibel. The leptin receptor mediates apparent autocrine regulation of leptin gene expression. Biochem. Biophys. Res. Commun. 240: 492-495, 1997[Medline].


Am J Physiol Endocrinol Metab 276(5):E836-E842
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society