Leptin responsiveness of juvenile rats: proof of leptin function within the physiological range

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1 11364 Journal of Physiology (2001), 530.1, pp Leptin responsiveness of juvenile rats: proof of leptin function within the physiological range Sandra Eiden, Gerald Preibisch* and Ingrid Schmidt Max-Planck-Institut für physiologische und klinische Forschung, W. G. Kerckhoff- Institut, Parkstraße 1, D Bad Nauheim and *Aventis Pharma, DG Metabolic Diseases, D Frankfurt, Germany (Received 6 July 2000; accepted after revision 15 September 2000) 1. To bridge the gap between studies demonstrating leptin s role in protecting fat stores when food is scarce and other studies demonstrating the effects of treatment with leptin at doses that increase plasma levels to values found in overfed animals, we investigated whether leptin serves an adipostatic function within the normal range of free-feeding lean animals, i.e. within the very small range of endogenous plasma levels at which no leptin resistance occurs. 2. For this purpose we applied recombinant leptin via mini-osmotic pumps to rats between 15 and 24 days of age and between 25 and 34 days of age and studied its dose-dependent effects on body mass and fat mass at plasma leptin concentrations extending down to the normal levels in lean animals. 3. Using percentage change of fat mass (relative to that of saline-treated littermates) as the measure, a linear dose response curve was found up to doses of 2 µg g _1 day _1, corresponding to plasma leptin concentrations between the normal physiological range and 50 ng ml _1. In 15- to 24-day-old animals, analysis of the correlation (r = _0.89) between individual plasma concentrations and the corresponding leptin-induced changes of body fat content for a range extending down towards zero (i.e. towards the average fat content of the controls) yielded a zero value of 3.1 ng ml _1, which was within the 2 4 ng ml _1 range of plasma leptin concentrations found in the control pups. Likewise, regression analysis for the data from the 25- to 34-day-old pups (r = _0.88), for which the control range was 1 3 ng ml _1, yielded a zero value of 1.9 ng ml _1. 4. We conclude that normal plasma leptin levels represent an adipostatic signal. Steady-state levels of plasma leptin in free-feeding lean animals thus provide a signal not only for protecting sufficiency but also for limiting increases of body fat stores. A signal produced by adipose tissue and signalling to the brain the total amount of fat stored in the body had been postulated by physiologists in the 1950s as the basis of systemic energy balance regulation (Kennedy, 1953). With the cloning of the ob gene in 1994 this regulatory concept turned into a known protein structure, a hormone, later named leptin (Zhang et al. 1994). Despite a huge number of leptin studies carried out since then, rather little is known about the effect of physiological changes in plasma leptin concentration, because most studies in wild-type animals demonstrated leptin effects only with pharmacological doses of recombinant leptin (Pelleymounter, 1997; Harris et al. 1998). Taken together with the observation that plasma leptin levels in untreated animals increase with increasing fat content, thus indicating leptin resistance at increased leptin levels, this favoured the hypothesis that plasma leptin concentrations might represent an effective regulatory signal only on the low side of the normal range (Ahima et al. 1996; Flier, 1998). Three recent studies on adult lean mice and rats with free access to food (Halaas et al. 1997; Harris et al. 1998; Ahima et al. 1999), using mini-osmotic pumps with the aim of determining dose response relationships for leptin in the physiological range, have arrived at contradictory conclusions about its role as an adipostatic signal in freefeeding lean animals. While the two studies using changes in body mass and food intake as indicators reported clear effects (Halaas et al. 1997; Ahima et al. 1999), the only study actually investigating the dose dependency of changes in body fat content failed to show an effect at low doses (Harris et al. 1998). Moreover, as pointed out by Ahima et al. (1999), the range of plasma leptin concentrations between 1.5- and 5-fold above normal, for which dose-dependent changes of body mass were

2 132 S. Eiden, G. Preibisch and I. Schmidt J. Physiol reported, corresponds to levels occurring spontaneously only in overfed and diet-induced obese animals, which are known to be leptin resistant. On the other hand, marked differences in food intake occurred during refeeding of starved animals, depending on whether or not their plasma leptin concentration had been experimentally maintained at its normal free-feeding level (Ahima et al. 1999). Thus, these studies emphasized rather than answered the question of whether leptin, apart from protecting body fat stores from becoming subnormal (Ahima et al. 1996; Flier 1998), also functions as a signal to limit body fat reserves on the high side. To clarify the role of changes in plasma leptin concentrations within the physiological range of normally fed animals, i.e. within the very small range of endogenous plasma leptin concentrations at which no leptin resistance occurs, we have exploited the high precision with which 2- to 5-week-old rat pups control their body fat content. By using regression analysis on the treatment effects of moderate to very low leptin doses, we were thus able to relate leptin treatmentinduced changes in body fat content extending down towards zero with the normal variations of plasma leptin concentrations in individual free-feeding animals. METHODS Animals As in our previous studies (Stehling et al. 1996; Kraeft et al. 1999), we used wild-type (+/+) Zucker rat (13M) pups reared in our colony at 22 C with lights on from to h. The pups in each litter were individually marked with subcutaneous injections of India ink on the first days of life. Pups that were treated from day 15 to 24 remained with their mothers throughout the study (12 litters), while pups treated from day 25 to 34 had been weaned at day 21 (4 litters). All procedures were carried out according to the German law for the protection of animals and were approved by the local veterinary control institution for animal care and use. Treatment protocol Recombinant His 6 -tagged murine leptin (17560 Da) produced as previously described (Müller et al. 1997) or phosphate-buffered saline (PBS) was applied via mini-osmotic pumps implanted under anaesthesia (see below). Average body masses before the start of the treatment were matched as closely as possible between that half of the litter receiving leptin and the sex-matched half serving as controls. Since the body fat content of untreated wild-type rat pups is closely correlated with body mass (Meierfrankenfeld et al. 1996), this helped to balance initial fat mass between treatment groups. Leptin was given in various doses, but pups belonging to the same litter were all treated with the same dose. Mini-osmotic pumps were implanted in the evening before the pups were 15 or 25 days old. The pumps were not incubated before they were implanted, in order that the rates of leptin delivery would become constant near the start of day 15 or 25. To determine plasma leptin concentrations at the beginning of the treatment period, two additional litters of the younger age group and one additional litter of the older group were implanted with mini-osmotic pumps and killed after 24 h. Because per-gram doses delivered by pumps running with constant delivery rates obviously decrease with growth, the average leptin dose per gram body mass throughout the experiment was, therefore, estimated from the pumping rate specified by the manufacturer and, assuming a roughly constant growth rate, the mean of the initial and final body masses. Implantation of pumps Mini-osmotic pumps (Alzet, Alza Corporation, Palo Alto, CA, USA) were implanted subcutaneously in the nape of the neck. In the five older litters type 2002 was used; in the younger litters the smaller type 1002 was used except in three litters where the larger pumps were necessary to deliver very high leptin doses. Pups were anaesthetized either with 30 mg g _1 Medetomidin plus 60 mg g _1 ketamine (I.M.) or with halothane in combination with N 2 O provided over an inhalation mask. During implantation of the mini-osmotic pumps, which on the average took 10 min or less, surgical anaesthesia was ascertained by testing for complete absence of any defence reflexes, and in addition, the skin was locally anaesthetized (lidocaine (lignocaine)). Normal mother litter interactions were observed in suckling-age pups, as soon as pups had completely recovered from anaesthesia, and normal food-intake behaviour was also observed in the weanlings. Pups (which will usually vocalize in response to painful skin stimuli) did not show any signs of discomfort after the local anaesthesia had worn off, even when the sutured skin wound was touched. Plasma collection and determination of body composition Pups were exposed to CO 2 for 30 s and then decapitated at the end of their daily light phase on the final day of treatment. Blood was collected in heparinized tubes on ice and centrifuged. Plasma aliquots were then stored at _80 C until leptin concentrations were determined. Plasma leptin concentrations were corrected for heparin dilution (determined by weighing). Carcass mass was determined after removing the stomach and intestines and emptying the bladder. Body composition water, fat and fat-free dry mass (FFDM) was evaluated by drying the carcass to constant weight and extracting the fat by using chloroform in a Soxhlet apparatus (Markewicz et al. 1993). Determination of plasma leptin Leptin concentrations were measured using a commercial murine radioimmunoassay (RIA) kit (Linco, St Charles, MO, USA). To allow for differences in antibody affinity, we corrected the kit-determined values by comparing them with defined amounts of recombinant His 6 -tagged murine leptin or with rat leptin standards (Linco). To obtain the necessary precision, samples were appropriately diluted so that measurements were carried out only in a small, particularly sensitive range of the binding curve, between 1 and 3 ng ml _1 for control animals and between 1 and 5 ng ml _1 for leptin-treated animals. For each animal we averaged the results of three to five measurements carried out in different RIAs (inter-assay variability 7%), with samples of littermates always measured within the same RIA. The measurement of three to five pool samples in the range between 1 and 3 ng ml _1 in each assay was used to determine the intra-assay variability (< 5%). Evaluation When evaluating the data on body composition, carcass mass and body mass at each age, we arranged the litters in groups treated with similar doses. For the evaluation of absolute values, as in previous studies, we took into account the considerable between-litter variability in growth (Truett et al. 1995; Meierfrankenfeld et al. 1996; Schwarzer et al. 1997), as well as sex-related differences in these variables, by considering a combined litter sex effect as the second factor (beside treatment) in two-way ANOVAs for each of these groups. This was done by simply assigning different litter numbers to male and female littermates. In this way, we could thus clearly separate the treatment effects from the highly significant

3 J. Physiol Leptin functions in the physiological range 133 Table 1. Differences in body mass and body composition between leptin-treated and control pups (PBS) after a 10 day treatment with various leptin doses Postnatal days Postnatal days Leptin PBS Leptin PBS Dose (µg g _1 day _1 ) 0.08 ~0.08 n Initial body mass (g) 27.9 ± ± ± ± 0.7 Final body mass (g) 53.5 ± ± ± 0.9 * 88.6 ± 0.9 Carcass mass (g) ± ± ± 0.81 * ± 0.90 Fat (g) 3.31 ± ± ± 0.09 *** 5.03 ± 0.10 FFDM (g) 9.47 ± ± ± ± 0.21 Water (g) ± ± ± ± 0.70 Dose (µg g _1 day _1 ) ~ n Initial body mass (g) 27.0 ± ± ± ± 0.6 Final body mass (g) 50.5 ± ± ± ± 1.7 Carcass mass (g) ± ± ± ± 1.30 Fat (g) 3.12 ± 0.11 *** 3.87 ± ± 0.34 * 3.05 ± 0.28 FFDM (g) 9.09 ± ± ± ± 0.21 Water (g) ± ± ± ± 0.87 Dose (µg g _1 day _1 ) ~2.0 n 7 10 Initial body mass (g) 26.2 ± ± 0.3 Final body mass (g) 44.3 ± 0.9 *** 50.5 ± 0.9 Carcass mass (g) ± 0.90 * ± 0.85 Fat (g) 1.22 ± 0.18 *** 3.45 ± 0.17 FFDM (g) 8.02 ± 0.21 * 8.83 ± 0.19 Water (g) ± 0.58 * ± 0.54 Least squares means ± S.E.M. of initial and final body mass, carcass mass and body composition data for 24- and 34-day-old leptin-treated (leptin) and control (PBS) pups after 10 day treatments with various leptin doses supplied by mini-osmotic pumps. n, number of pups treated with leptin/pbs. * P < 0.05, *** P < 0.001, all other P > litter sex effect. Mean values are consequently presented as least square means (± S.E.M.). In addition, to evaluate the absolute amounts of fat, FFDM and water, we also determined the changes in these quantities after leptin treatment by calculating for each pup the percentage difference from the mean value for the control littermates. As this procedure normalizes interlitter differences, only sex and treatment had to be considered as factors in the statistical analysis of these data. The dose dependence of leptin-induced changes in the body fat content of pups from different litters was also evaluated by regression analysis, another method allowing for the inter-litter differences in growth (Meierfrankenfeld et al. 1996; Kraeft et al. 1999). RESULTS Effects of leptin on body composition of pre- and postweanling rat pups Giving pups about 0.3 µg g _1 day _1 of recombinant leptin via mini-osmotic pumps from postnatal day 15 to 24 reduced their body fat content 20% below that of their control littermates without significantly changing their total carcass mass, FFDM, or water content (Table 1). This dose is slightly below the lowest dose known to produce a clear effect on the body fat content of 7- to 16-day-old pups treated with two daily injections (Stehling et al. 1997). A 7 times higher dose (2 µg g _1 day _1 ), well within the range of doses frequently used in studies of adult animals (Pelleymounter, 1997; Harris et al. 1998) resulted in a body fat content 60% below that of the control littermates and also produced significant, albeit small, changes of lean body mass. Pups treated from postnatal day 25 to 34 with 0.08 µg g _1 day _1, a dose that had no effect on the younger pups, developed a body fat content 30% below that of their control littermates without significant changes of FFDM (Table 1). A body fat content 60% below that of control pups was produced by a leptin dose of only 0.3 µg g _1 day _1. Because analysis of body composition shows that leptininduced changes in total body fat content of about 30% are accompanied by changes in FFDM of only 2%, it is often assumed that changes in body mass observed after leptin treatment are solely due to changes in body fat content. Therefore it is important to note that roughly 10 20% of the change in total body mass was due to changes in stomach and gut contents (Table 2), and only half of the remaining change was due to changes in body

4 134 S. Eiden, G. Preibisch and I. Schmidt J. Physiol Table 2. Changes in gut contents and body composition resulting after a 10-day leptin-infusion period Postnatal days Postnatal days Difference Body mass Difference Body mass (leptin _ PBS) change (leptin _ PBS) change (g) (%) (g) (%) ~0.30 µg g _1 day _1 leptin ~0.08 µg g _1 day _1 leptin Body mass _ _ Gut contents _ _ Fat _ _ FFDM _ _ Water _ _ ~2.0 µg g _1 day _1 leptin ~0.30 µg g _1 day _1 leptin Body mass _ _ Gut contents _ _ Fat _ _ FFDM _ _ Water _ _ Shown are the absolute changes in final body mass, stomach and gut content (intestinal filling), fat mass, fat-free dry-mass (FFDM) and body water expressed as differences between leptin-treated (leptin) and control pups (PBS) and the changes in these variables expressed as a percentage of the total difference in final body mass after a 10-day infusion period. Data derived from the averages given in Table 1. fat content; the rest was due to changes in lean body mass. Changes in body water content make particularly large contributions (about 30%) to the total change in body mass, while changes in FFDM contribute only about 10%. Correlation between plasma leptin levels and body fat content in control pups Figure 1 shows that the logarithm of the plasma leptin levels of 24- and 34-day-old control pups was closely correlated with body fat content. At any given fat content, the leptin levels in the younger pups were higher than those in the older pups (P < for comparison of parallel regression lines in Fig. 1A), but because, at the same absolute fat mass, the relative body fat content of the older and bigger pups was much smaller, this apparently different relation between body fat content and leptin level disappeared when plasma leptin levels were plotted against the percentage of body fat (Fig. 1B; P > 0.05 for comparison of parallel regression lines). No sex difference in the relation between body fat content and plasma leptin levels could be detected in either age group. In the 24-day-old control pups, the average plasma leptin concentration was 2.8 ± 0.1 ng ml _1, the average body fat mass was 3.6 ± 0.1 g, and the percentage body fat was 8.1 ± 0.2%. The corresponding values for the 34-day-old control pups were 1.9 ± 0.2 ng ml _1, 4.3 ± 0.3 g and 6.3 ± 0.2%. Correlation between plasma leptin levels and body fat content in leptin-treated pups Final plasma leptin levels of pups given leptin via miniosmotic pumps from day 15 to 24 increased linearly over a wide range of doses calculated for the last 24 h of the treatment period (Fig. 2). Separate calculation of regression lines for the lower ( physiological ) range (below 10 ng ml _1, r = 0.78), and for the whole range of doses investigated (up to 60 ng ml _1, r = 0.93) yielded no significant difference (P > 0.05 for difference of parallel regression lines). The plasma leptin concentrations of 34-day-old pups fitted well onto the regression line calculated for the younger pups. Moreover, even data from adult leptin-treated rats, derived from a study by Ahima et al. (1999), matched the regression line. Because pups are rapidly growing during the 10 day treatment periods, the doses applied by pumps running with a constant rate decrease by about 50% throughout the treatment period. The temporal mean plasma concentration during the entire treatment period can, however, be estimated from the correlation between the final plasma leptin concentration and the final dose, if we calculate the temporal mean body mass assuming an approximately linear growth rate and a constant relation between plasma leptin level and infused doses. For both age groups, the percentage change in body fat content for the leptin-treated pups (relative to the body fat content of the control pups) is closely correlated with the logarithm of the temporal mean plasma leptin concentration (Fig. 3A). At the same plasma leptin concentration, the older pups showed greater leptin-induced decreases in body fat content than the younger pups (P < for difference of regression lines), clearly showing the higher responsiveness of the older pups. Final body mass, in contrast, is much less closely correlated with the plasma leptin concentration and regression analysis does not yield a significant difference between the regression lines for the two age groups (Fig. 3B). However, re-evaluating

5 J. Physiol Leptin functions in the physiological range 135 previously published data on the effect of leptin on body mass in adult rats (Ahima et al. 1999) in the same manner strongly suggests that leptin sensitivity increases and remains high in young adults. Do changes in leptin pharmacokinetics distort the correlation between leptin infusion rate and plasma leptin concentration? Even though the final data were gathered at a time when the implanted pumps were still infusing leptin at the rate specified by the manufacturer, the relation between infusion rate (per gram body mass) and plasma leptin level might change during development, if the rate at which leptin is degraded or eliminated from the blood stream varies either with age or with the duration of obtaining exogenous leptin at high rates. If so, the values calculated above for the temporal mean plasma concentration during the entire treatment period would be incorrect. We therefore implanted mini-osmotic pumps in a couple of 14- and 24-day-old litters and killed the pups 24 h later in order to measure their plasma leptin concentrations. The mean value for the pups treated only throughout day 25 lay within the 95% confidence interval of the regression line calculated for the pups that had been treated from days 15 to 24 (Fig. 2B), indicating that the plasma clearance rate in the older pups did not change during treatment. The mean value obtained for the pups treated only throughout day 15 lay slightly above the 95% confidence interval for day 24, indicating that the clearance rate in the youngest pups might have been somewhat lower. The deviation is so small, however, that the actual temporal mean plasma concentrations Figure 1. Plasma leptin concentration (log scale) in 24- and 34-day-old control pups as a function of body fat mass (A) and percentage body fat (B) Continuous lines show regressions for the younger pups (circles, r = 0.64 in A; r = 0.59 in B) and dashed lines show regressions for the older pups (diamonds, r = 0.86 in A; r = 0.73 in B). Filled symbols with error bars show arithmetic means ± S.E.M. for each age group. Symbols marked with dots represent females; no sex difference is evident. Figure 2. Plasma leptin concentration as a function of final leptin dose after a 10 day treatment via mini-osmotic pumps Data of 24-day-old (0) and 34-day-old pups (2) are shown for comparison. The average plasma leptin concentrations in the control pups of the same age are indicated by continuous (day 24) and dashed (day 34) lines. A, full dose range. B, enlargement of the lower ( physiological ) dose range. Open symbols (not included in the regression analysis) show data from 24 h infusion experiments on day 15 (1) and day 25 (3). Bold open symbols with error bars show the corresponding means ± S.E.M. Large crosses show leptin levels after a 14 day treatment of adult rats derived from Ahima et al. (1999).

6 136 S. Eiden, G. Preibisch and I. Schmidt J. Physiol over the 10 day treatment period in the group of 15- to 24-day-old pups would be only slightly higher than those calculated from the correlation between the final plasma leptin concentration and the dose infused on the last day of the experiment. Because so many assumptions are required in calculating the temporal mean of the plasma leptin concentration, we also evaluated the correlation between the directly measured final plasma leptin concentration and percentage change in body fat (not shown). In this case, the regression lines for the younger and older pups (r = _0.88 and r = _0.62, respectively) were significantly shifted (P < 0.001) in parallel with the line for the older pups, having a lower y-intercept. The corresponding x-axis intercepts for the two age groups were 2.2 and 0.7 ng ml _1. Since the per-gram dose infused into the growing pups on the final day must have been lower than the average dose infused throughout the treatment period, these values also correspond quite well to the average normal plasma leptin values of 2.8 and 1.9 ng ml _1 found for 24- and 34-day-old control pups. DISCUSSION Leptin functions within the physiological range The salient point of this study is that we were able to correlate individual plasma leptin concentrations with the corresponding leptin-induced changes in body fat content extending down towards zero (i.e. towards the average body fat content of the controls). Thus we were able to demonstrate that leptin provides a regulatory signal for the control of body energy stores when it is present at plasma concentrations within the normal range of freely feeding lean animals. The regression between the leptin-induced changes in body fat content and temporal mean plasma leptin concentration determined in this study (see Fig. 3) yielded x-intercepts of 3.1 ng ml _1 plasma leptin for the younger pups and 1.9 ng ml _1 for the older pups. These values are surprisingly similar to the average plasma leptin levels of untreated pups of the same age: 2.7 ± 0.2 ng ml _1 for 16-day-old pups (C. Hufnagel & I. Schmidt, unpublished observations), 2.8 ± 0.1 ng ml _1 for 24-day-old pups, and 1.9 ± 0.2 ng ml _1 for 34-day-old pups (Fig. 1). These Figure 3. Differences ( ) in fat mass (A) and body mass (B) between leptin-treated and control pups as a function of the estimated temporal mean of plasma leptin concentration (log scale) during the 10 day treatment Data of 24-day-old (0) and 34-day-old (2) pups are shown as the percentage by which data of leptintreated pups differ from the mean values for control pups. The estimate of the temporal mean plasma leptin concentration is based on the assumption of linear growth, and that the relation between plasma leptin concentration and infused dose was constant throughout the experiment (see Fig. 1). Continuous lines show regressions for the younger pups (r = _0.89 in A; r = _0.56 in B) and dashed lines show regressions for the older pups (r = _0.88 in A; r = _0.36 in B). Symbols marked with dots represent females; no sex difference is evident. Large crosses show for comparison data derived from Ahima et al. (1999) for adult leptin-treated and control rats for which only body mass but not body fat was determined at the end of a 14 day treatment period. Figure 4. Age-dependent effect of four different leptin doses on the body fat mass of rat pups Shown for the leptin-treated pups at each age are the least square means + S.E.M. for the percentage differences from the mean of the control littermates after a treatment period of 7 days (in the first postnatal week) or 10 days (in the older pups). Doses are given in µg g _1 day 1. Data from 1- and 2-weekold pups have been derived from Kraeft et al. (1999) and from Stehling et al. (1997).

7 J. Physiol Leptin functions in the physiological range 137 findings bridge the gap between previous studies clearly demonstrating leptin functions in starving animals, on the one hand (Ahima et al. 1996, 1999), and others demonstrating leptin functions in response to pathophysiological plasma leptin concentrations between 1.5- to 5-fold above normal, on the other hand (Halaas et al. 1997; Ahima et al. 1999). As previously pointed out (Ahima et al. 1999), increases in plasma leptin to levels 1.5- to 5-fold above normal, although often claimed as physiological, would spontaneously occur only in overfed animals at the onset of diet-induced obesity, a state known to be associated with leptin resistance (Vanheek et al. 1997; Lin et al. 2000). Plasma concentrations in 3- to 5-week-old wild-type Zucker rats (see Fig. 1) and Wistar rats (Plagemann et al. 1999b) reared by their mothers in litters of normal size vary only between 1 and 4 ng ml _1. In contrast, plasma leptin concentrations in Wistar weanlings that have been overfed by rearing in small litters vary between 4 and 20 ng ml _1, and these animals have correspondingly increased body fat stores and have become completely leptin resistant (Plagemann et al. 1999b; Schmidt et al. 2000). This underlines the importance, when making statements about the working range of leptin as a physiological adipostatic signal, of differentiating between variations in leptin concentrations within the normal range in freely feeding lean animals and pathophysiological increases in leptin concentrations in overfed, leptin-resistant animals at the onset of obesity. Thus, positive correlations between increased body fat content and leptin levels should not be taken as evidence against a role for normal plasma concentrations of leptin for the regulation of body fat content, unless an experimental increase in leptin over extended periods can be shown to trigger leptin resistance even if body fat content is going down rather than up, as it does during the development of diet-induced obesity associated with hyperleptinaemia and leptin resistance. Depending on the long-term level of food availability (or the corresponding size of endogenous fat stores) animals will recruit different effector mechanisms in response to the same doses of recombinant leptin (Döring et al. 1998a). Specifically, in semi-starved mice, chronically restricted to 80% of normal food intake, leptin treatment changes metabolic rate, but is not able to suppress food intake, whereas the opposite is the case in free-feeding mice. This observation is paralleled by reports of differing effects of leptin treatment on hypothalamic expression of the anorectic peptides a-msh and CART in starved and freefeeding rats, whereas the leptin-induced changes in the orexigenic peptide NPY were similar in both physiological conditions (Ahima et al. 1999). Interestingly, however, despite these differences in neuropeptide expression and recruited effector mechanisms, leptin treatment in semistarved mice and mice fed ad libitum caused the same percentage changes in body fat content (Döring et al. 1998b), a finding which strongly supports the concept of leptin as a signal controlling total body fat content not only on the low but also on the high side of the normal range. While it has been rightly argued that overfeeding is a problem that has never emerged in the course of evolution (Ahima et al. 1996; Flier, 1998), an important biological task has been not only maintaining body fat just above a critically low level, but also regulating fat stores at a certain level above minimum. For instance many species show marked, closely controlled annual cycles of adiposity which would demand information provided by an adipostatic signal (Steinlechner et al. 1983; Klingenspor et al. 2000). Even for juvenile animals, it is important to be protected not only against critically low body fat stores, but also against abnormally high fat stores during suckling age because juvenile obesity might have lifelong adverse consequences even if food availability after weaning is normal (Plagemann, 1999a). Moreover, modifying food intake not only according to short-term signals but also according to an integrated adipostatic signal would enable an animal to rate the importance of immediate food intake in comparison to competing behavioral demands, a task of eminent evolutionary relevance for juvenile as well as adult animals. Developmental changes in leptin responsiveness Taken together, the results of this study in 2- to 5-weekold pups and of the previous studies on 1- to 2-week-old rat pups (Kraeft et al. 1999; Stehling et al. 1996, 1997) provide an overview of the changes in leptin responsiveness from birth through the suckling, weaning, and the early postweaning periods (Fig. 4). As the present study shows, leptin responsiveness increases markedly during the third week of life and becomes even more pronounced after weaning. This is particularily interesting, because the percentage body fat content and plasma leptin concentrations in 3-week-old rats are similar to those found in 3-month-old rats (Ahima et al. 1999; authors unpublished results). After weaning, the reduction in body fat content (relative to the control littermates) caused by treatment with a moderate dose of leptin is nearly four times the reduction caused by the same leptin treatment during the second postnatal week (Stehling et al. 1996, 1997). Further, comparison of the effects caused by leptin treatment on the body mass of adult rats suggests that the high level of sensitivity at weaning is maintained into young adulthood (see Fig. 3). However, because large percentage changes in body fat content cause only small changes in the total body mass of wild-type animals with low body fat content, body mass is not a very reliable indicator of the magnitude of leptin effects (Table 2; Stehling et al. 1997; Döring et al. 1998a), and thus, quantitative information about the effect of leptin on body fat content will be needed to confirm this suggestion for adult animals. Whether the changes in leptin responsiveness from suckling age through the

8 138 S. Eiden, G. Preibisch and I. Schmidt J. Physiol weaning process are due to developmental changes in the sensitivity of leptin receptors and/or in the recruited effector mechanisms also awaits further studies. Precision of plasma leptin concentration measurements It is rarely mentioned that the plasma leptin levels reported in different studies for rodents under similar physiological conditions vary widely (Blum et al. 1997). But because the range of plasma leptin concentrations occurring in animals which are not leptin resistant is very narrow, low levels of plasma leptin must be determined with appropriate precision. To obtain sufficiently reliable determinations of plasma leptin concentrations in this study, the restriction of measurements to a small, highly sensitive range of the RIA curve and averaging of multiple measurements were essential. If high precision is demanded, differences in biological activity between recombinant murine leptin and native leptin in the rat have to be taken into consideration as well as the possibility that RIA values, reflecting immunoreactivity, do not provide reliable information about the biological activity. Concerning potential differences in the detection of native and recombinant leptin by our RIA, the y-intercept of 2.2 ng ml _1, yielded by the regression between the leptin doses and the resulting estimated plasma leptin concentrations in pups treated from day 15 to 24, approached the 2.8 ng ml _1 mean leptin concentration found in untreated control pups of the same age (see Fig. 2) closely enough to demonstrate that this is not the case. Concerning potential errors in estimating the biological activity of the recombinant leptin, on the other hand, it has to be considered that potential errors between the RIA estimates and the true biological activity will affect only the slopes of the dose response curves and will approach zero when infused doses become so low that plasma leptin levels approach the normal endogenous level (see Fig. 3). These errors, thus, do not affect the conclusions drawn from the extrapolation of the dose response curves towards zero. Role of leptin in an adipostatic hypothesis Body fat content might normally be controlled by feedforward signals (e.g. the well-known set of signals starting and stopping food intake (Novin, 1983; Kalra et al. 1999). But when these feedforward responses are unable to prevent changes in body fat content, a leptin signal proportional to body fat mass would provide a steady-state negative feedback signal as long as animals are not leptin resistant. For the low range of plasma leptin concentrations seen in starving animals this has, indeed, been convincingly demonstrated (Ahima et al. 1999). A first experimental hint that normal leptin levels in lean animals also constantly contribute to an adipostatic signal limiting increases in body fat content was provided by the observation that food intake increases in response to I.C.V. application of leptin antibodies, suggesting a tonic inhibitory influence of normal local leptin concentrations on food intake (Brunner et al. 1997). Our finding of a log-linear dose response curve extending from very high plasma leptin concentrations all the way down to the normal range further supports the hypothesis that normal variations in plasma leptin levels represent a small proportional signal important for the steady-state feedback control of body fat content in free-feeding lean animals. AHIMA, R. S., KELLY, J., ELMQUIST, J. K. & FLIER, J. S. (1999). 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