Mechanisms for LEPR-mediated regulation of leptin expression in brown and white adipocytes in rat pups

YIYING ZHANG1, CARMEN HUFNAGEL2, SANDRA EIDEN2, KAI-YING GUO1, PATRICIA A. DIAZ1, RUDOLPH L. LEIBEL1 and INGRID SCHMIDT2

1 Division of Molecular Genetics, Department of Pediatrics, Columbia University College of Physicians and Surgeons, New York, New York 10032
2 Max-Planck-Institut, fuer physiologische und klinische Forschung, W. G. Kerckhoff-Institut, D-61231 Bad Nauheim, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the underlying mechanisms for leptin receptor (LEPR)-mediated regulation of leptin gene (Lep) expression in brown (BAT) and white (WAT) adipose tissue and resultant effects on plasma leptin concentrations (plasma-LEP), we examined effects of sympathetic nervous system (SNS) activity, caloric balance, and body fat content on leptin mRNA levels in BAT and WAT in 10-day-old rat pups segregating for Leprfa. In mother-reared pups, Lep mRNA levels were fa/fa > +/fa = +/+ in BAT and was fa/fa > +/fa > +/+ in WAT. The genotype effects on Lep expression in BAT and plasma-LEP were virtually eliminated when the differences in SNS activity between fa/fa and +/fa pups were equalized by artificial rearing of pups under thermoneutral conditions with or without oral norepinephrine (NE) administration. NE administration alone had little effect on the Leprfa-dependent stratification of Lep expression in WAT. BAT-Lep mRNA was the main determinant of plasma-LEP. Metabolic rate, a surrogate indicator of SNS activity, explained 87% of the variation in BAT-Lep mRNA (R2 = 0.93), whereas caloric balance (40%) and body fat mass (6%) accounted for most of the variation in WAT-Lep mRNA (R2 = 0.53). We conclude that feedback regulation of Lep expression in BAT is primarily via central nervous system-mediated effects of leptin on SNS activity, whereas the control of leptin expression in WAT is more likely via mechanisms not directly dependent on SNS activity.

energy metabolism; obesity; sympathetic nervous system; leptin receptor; adipose tissue


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LEPTIN, a hormone synthesized predominantly in adipose tissue, plays an important role in energy homeostasis (55). Plasma leptin concentrations (plasma-LEP) are highly correlated with body fat mass in both humans and rodents (18, 34, 37) and may provide the central nervous system (CNS) with a signal regarding the size of fat stores and the rate of substrate flux through these stores (18, 52). The mechanisms underlying the regulation of leptin gene (Lep) expression in white (WAT) and brown (BAT) adipose tissues are not well understood.

We have previously shown that leptin mRNA levels in inguinal WAT, body fat mass, and plasma-LEP per unit of fat mass are increased in 10-day-old Zucker rat pups in proportion to gene dosage for the functionally inactive leptin receptor, Leprfa (54). These effects of Leprfa are independent of plasma insulin concentrations (54), suggesting that leptin expression in adipose tissue is subject to negative feedback regulation that is mediated by the leptin receptor. Similar mutation dose-dependent increases in body fat content and plasma-LEP relative to fat mass were also observed in adult mice segregating for Leprdb (8). These effects of gene dosage for Lepr mutations could be conveyed via autocrine and/or CNS-mediated effects of leptin action (5, 12, 38, 56).

Decreased sympathetic nervous system (SNS) tone is a major characteristic of animals with defects in the leptin axis (10, 53). SNS activity is an important regulator of leptin gene expression in adipocytes. Sympathetic activation by cold exposure, or in vivo or in vitro administration of ß3-adrenergic receptor agonists, inhibits leptin mRNA expression in BAT and WAT in rodents (13, 17, 29, 48). Thus SNS activity could play an important role in mediating leptin’s feedback regulation of its own expression in adipose tissue. Leptin also directly inhibits its own mRNA levels in adipose tissue (56) and exerts direct effects on lipolysis and glucose utilization in adipocytes in vitro (19, 40, 42). Thus adipocyte leptin receptor (LEPR) may mediate direct effects on Lep expression and/or indirect effects through effects on metabolite flux in adipocytes (19, 29, 42, 52, 56).

BAT is an important thermogenic organ not only in cold-adapted rodents but also in newborns of most species, including human infants. In rats it constitutes the only adipose tissue visible at birth (14). Both BAT and WAT contain triglycerides and express many common adipocyte-specific genes, such as aP2, Lep, PPAR-{gamma} (29, 42, 46). However, BAT differs from WAT most significantly in its thermogenic capacity and extensive sympathetic innervation (2, 3, 41). These differences in function, and in the extent of SNS innervation between BAT and WAT, make them ideal tissues in which to examine the role of SNS activity in conveying LEPR-mediated suppression of Lep gene expression in adipocytes. Although Lep gene expression is very low in BAT of adult animals (9), its expression is more abundant in BAT of newborn animals and suckling pups (14, 15).

Artificial rearing of rat pups under thermoneutral conditions with or without norepinephrine (NE) administration has been used to assess SNS-mediated effects on body fat content in fa/fa and +/fa rat pups (20, 21, 26). Under normal rearing conditions (pups suckled by dam), pups are intermittently exposed to cold loads whenever the mother leaves the nest, resulting in intermittent activation of the SNS (35, 36). Activation of thermoregulatory thermogenesis under such circumstances is much smaller in fa/fa pups than in +/fa and +/+ pups (32, 36). Artificial rearing under thermoneutral conditions prevents such transient stimulation of sympathetic tone, leading to a decreased but equal sympathetic tone in fa/fa and +/fa pups as indicated by identical rate of energy expenditure (20, 26). Artificial rearing under thermoneutral conditions with synthetic milk supplemented with NE, an SNS effector, mimics continuous activation of SNS in both fa/fa and +/fa pups and results in the same increased "effective SNS tone" in both fa/fa and +/fa pups (21). In addition, artificial rearing allows experimental control of caloric intake, enabling manipulation of metabolic flux and energy storage independently from energy expenditure in pups.

To investigate the role of SNS activity and metabolic flux and/or energy storage in mediating the feedback regulation of leptin on its own gene expression in adipocytes, we examined the effects of Leprfa gene dosage on leptin mRNA levels in BAT and WAT and plasma-LEP in 10-day-old rat pups under normal rearing conditions (mother rearing) and various artificial rearing conditions that affect SNS activity and caloric balance. In this model, which permits their experimental isolation, we compared the effects of differences in SNS activity, caloric balance, and body fat content on Lep mRNA expression in BAT and WAT and assessed their respective contributions to circulating leptin concentrations.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Experimental animals.
We used animals from the outbred Zucker rat colony at the W. G. Kerckhoff-Institut. This colony was founded in 1985 with breeding stock donated by Harry Carlisle of the University of California, Santa Barbara. Litter size is usually between 7 and 12 pups. To maximize the number of pups available for genotype-related intralitter comparisons, we mainly used fa/fa x +/fa, or +/fa x +/+ matings that produced only two genotypes in each litter. Twelve litters of 10-day-old mother-reared pups were used in this study. In five litters, only WAT and BAT samples were obtained for determination of Lep, Ucp1, and aP2 mRNA levels (n = 11 fa/fa, 17 +/fa, and 13 +/+). In another seven litters (litters 1–7, see Fig. 2), plasma leptin and body fat content were determined (n = 15 fa/fa, 33 +/fa, and 13 +/+). Lep mRNA levels in WAT and BAT samples were also determined in three of these seven litters (litters 1–3, see Fig. 2) (n = 8 +/fa and 10 +/+). For all artificially reared pups (litters 8–16, see Fig. 2), plasma-LEP, body fat content, and WAT-Lep mRNA and BAT-Lep mRNA were determined (sample size for each rearing condition is given below).



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Fig. 2. Scatter plots of body mass (A), mass-specific metabolic rate (MS-MR) during the last 24-h of life (B), percent body fat (C), and plasma-LEP (D), for artificially reared pups, +/+ (circles), +/fa (triangles), fa/fa (squares), grouped by litter. Rearing conditions are indicated above the plots. Days refer to the duration of the artificially rearing. FatHI and FatLO refer to high and low final body fat content obtained by manipulation of milk feeding rates, and NE refers to oral norepinephrine administration. Percent body fat and plasma-LEP from mother-reared pups were included as a control (litters 1–7).

 
General conditions for mother rearing and artificial rearing.
For mother rearing, pups and their dams were caged together and maintained at 22°C in a 12:12-h light-dark cycle until pups were 10 days old. Artificial rearing for a period of 6 or 2 days was carried out in batches of 10 pups as previously described (20, 21, 26, 31). Briefly, pups were kept individually in plastic containers floating in a temperature-controlled water bath and were continuously fed (via intra-esophageal catheters) a synthetic rat milk (8.6 kJ/ml) at rates of 2 ml/day on postnatal day 4, which was gradually increased to 3–5 ml/day on postnatal day 10/11. Feeding rates were identical for pups from the same litter but varied among litters. Care was taken that feeding rate did not exceed the rate of stomach emptying (visually monitored), which varies according to experimental conditions. Core temperature (Tc) of all 10 pups and oxygen consumption of 7 individual pups from each litter were continuously measured as previously described (20, 26, 30). A mass flow meter (Tylan) and electrochemical oxygen analyzer (Ametek, S-3A/II) in an open-flow system were used to successively measure, for 5 min once every 40 min, the flow rate and oxygen content of the dry air aspirated from each of seven animal chambers and one reference chamber. Water manometers were used for adjusting and monitoring the pressure in each of the aspiration lines to ensure that the airflow during the interval between the measurements was the same as that during the measurement. The metabolic rate (MR) was calculated assuming a respiratory quotient (RQ) of 0.75 and using a caloric equivalent of 0.33 W per ml O2 per min and a conversion factor of 20.0 kJ/liter O2 (26). For those pups in which only Tc was recorded, mass-specific MR (MS-MR) during the final 24-h of life was estimated based on the close correlation between these two parameters in 10-day-old pups (26, 33, 44, 45). Because the 24-h means of MR for all measured animals in each of the thermoneutrally reared litters differed by not more than 2 W/kg, a negligible error was introduced by using the litter average for those animals in which gas exchange had not been studied (see Fig. 2A). For the cold-reared and NE-treated litters, the correlation between 24-h means of Tc and MS-MR was determined for each of the litters (0.99 > r >0.85, 7 pups in each litter). The MR of the pups whose oxygen consumption had not been measured was estimated based on their Tc and the correlation between Tc and MR of their littermates.

Definition and calculation of metabolic parameters.
1) Total daily energy expenditure (MR) was determined using a conversion factor of 20 kJ/liter O2 (26, 32). For those pups in which only Tc was recorded, MR during the final 24-h of life was extrapolated using regressions of MR against Tc of their littermates based on the close correlation between these two parameters in 10-day-old pups (33, 44, 45). 2) MS-MR is defined as total daily energy expenditure/body mass; MS-MR for artificially reared 10-day-old pups under thermoneutral conditions is 8–9 W/kg (20, 21, 26). 3) Caloric balance is defined as the difference between milk calories fed and energy expenditure (MR) during the last 24 h of life.

Specific conditions for artificial rearing.
1) To artificially rear pups under cold loads similar to those experienced during normal rearing conditions, ambient temperature for litters 8 and 9 (n = 4 fa/fa, 7 +/fa, and 6 +/+) was adjusted so that the Tc of the +/fa littermates was in the range observed in mother-reared +/+ and +/fa pups (31, 35, 36, 39), causing MS-MR during the last 24 h of life to be about 70% above that of thermoneutrally reared pups of this age (20, 21, 26). All pups in these litters were fed at the maximal feeding rate compatible with the stomach emptying of the hypothermic fa/fa pups. One of these cold-reared litters (litter 8) had higher fat-free dry mass (FFDM) than predicted, indicating that the pups might have been a day older than expected. The data from this litter were included in all scatter plots (Figs. 2 and Go4), but not in any of the statistical analyses. 2) For thermoneutral rearing without NE treatment (litters 10–13, n = 23 fa/fa, 13 +/fa, and 5 +/+), the ambient temperature was adjusted to keep Tc slightly above 37°C, i.e., at the temperature at which the average daily MR was at its thermoneutral value (21, 31). Feeding rate was maintained at a rate similar to that in the cold-reared litters. 3) For thermoneutral rearing with NE treatment (litters 14–16, n = 13 fa/fa, and 13 +/fa), oral NE treatment under thermoneutral conditions was carried out exactly as previously described (21). In short, pups were started on artificial rearing with milk without NE on postnatal day 4. Starting at 5 days of age, pups were fed milk containing NE at concentrations that were identical for all pups in one litter. The dose of NE was adjusted twice daily to gradually increase MS-MR from its thermoneutral level on postnatal day 4 to about 100% above this level on postnatal day 10 or 11. Doses between 250 and 1,000 µg·kg-1·h-1 of free base were needed to obtain this effect. Ambient temperature was gradually decreased as necessary to maintain Tc between 37 and 39°C. To distinguish between the effects of variation in MR and variation in caloric balance and/or body fat content on leptin gene expression, we varied the milk feeding rates among the three NE-treated litters while keeping their MS-MR at similarly high levels. In two of these litters (litters 14 and 15), milk feeding rate throughout artificial rearing was maintained at the highest level compatible with the rate of stomach emptying, producing animals with a higher final body fat content (FatHI, see Figs. 2 and 3). In litter 16, milk feeding rate was kept at the lowest rate compatible with a normal growth rate (an average weight gain of 0.7 g/d), producing animals with a lower final body fat content (FatLO, see Fig. 2 and 3).



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Fig. 3. Least square means ± SE of leptin mRNA levels in WAT (A) and in BAT (B) of artificially reared +/+ (solid bars), +/fa (hatched bars), fa/fa (open bars) pups. Rearing conditions are indicated above the plots. Days refer to the duration of the artificially rearing. Data from mother-reared pups were included as a control. Effects of genotype at Lepr were determined by two-way ANOVA with litter (when more than one litter was analyzed) and genotype as grouping factors for each treatment group. Statistical significance was denoted as aP < 0.001, fa/fa vs. +/fa; bP < 0.01, fa/fa vs. +/fa; and cP < 0.001, +/fa vs. +/+.

 


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Fig. 4. Correlations between plasma-LEP and leptin mRNA levels in WAT (A) and BAT (B) in artificially reared pups. Logarithmic scales are used for both plasma-LEP and leptin mRNA levels in WAT or BAT. Symbols for genotypes at Lepr are: +/+ (circles), +/fa (triangles), fa/fa (squares). Symbols for rearing conditions are: thermoneutral rearing for 2 days (open symbols with black dots); thermoneutral rearing for 6 days (open symbols with crosses); cold reared, litter 8 (solid symbols with crosses); cold reared, litter 9 (solid symbols with white dots); NE treated with high final body fat content (small solid symbols); NE treated with low final body fat content (small open symbols). The correlation coefficients (r) of WAT-Lep and BAT-Lep mRNA with plasma-LEP in all artificially reared pups with plasma-LEP below 10 ng/ml (excluding cold-reared litter 8) were 0.39 and 0.78, respectively. The oversized symbols (larger than both the small and large symbols) represent the least square means of mother-reared pups after control for litter effects.

 
Tissue and plasma sample collection and determination of body composition.
Pups were anesthetized for 30 s by CO2 and decapitated. Trunk blood was collected on ice in tubes containing heparin as anticoagulant. Sample dilution was determined by weighing, and concentrations were appropriately corrected. Plasma was collected after centrifugation and stored at -80°C. Interscapular BAT (~60 mg) and a 10–20 mg sample of inguinal WAT were rapidly dissected and frozen immediately in liquid nitrogen. Total body fat and FFDM of carcasses were determined by drying the carcasses at 75°C to a constant weight, followed by whole body chloroform extraction as previously described (20, 26). The error introduced in determination of total body fat content in litters in which tissue samples were removed was ~3% of total body fat, and this measure was corrected accordingly.

Determination of genotype at Lepr.
Genotype was determined by the presence of a novel MspI site introduced by the Leprfa mutation. Genomic DNA isolated from tail clips was amplified by PCR using primers flanking the Leprfa mutation followed by MspI restriction digestion of the PCR products and gel electrophoresis analysis (7).

Determination of plasma-LEP.
Plasma-LEP were determined with a commercial murine leptin radioimmunoassay (RIA) kit (Linco, St. Charles, MO) and corrected against repeated measurements of a rat leptin standard (Linco) within the physiological range. In our hands, this method yielded more reproducible results, particularly in the lower range of plasma-LEP, than commercially available rat leptin RIA kits. Two or three aliquots of each plasma sample were assayed. The intra-assay and interassay variation was <5% and <10%, respectively.

Quantitative RT-PCR.
Total RNA from adipose tissue was extracted using TRIzol reagent (GIBCO; Life Technologies, Bethesda, MD). About 1–5 µg of total RNA was reverse-transcribed into cDNA using random hexamer and Moloney murine leukemia virus reverse transcriptase. Complementary DNA was amplified by PCR using specific primers for Lep, aP2, or Ucp1 in combination with primers for ß-actin (as an internal control) as previously described (54). Primers for Lep were 5'-TGACACCAAAACCCTCATCA-3' (forward) and 5'-AGCCCAGGAATGAAGTCCA-3' (reverse), for Ucp1 were 5'-GGTGAGTTCGACAACTTCCG-3' (forward) and 5'-GGTGATGGTCCCTAAGACACC-3' (reverse), for aP2 were 5'-GACGACAGGAAGGTGAAGAGC-3' (forward) and 5'-GCCTTTCATAACACACATTCCACC-3' (reverse), and for ß-actin were 5'-AGGCCCAGAGCAAGAGAG-3' (forward) and 5'-GGGTGTTGAAGGTCTCAAAC-3' (reverse). [32P]dCTP-labeled PCR products were visualized and quantified using a PhosphoImager (Molecular Dynamics). Message RNA levels for Lep, aP2, and Ucp1 were expressed as a ratio to ß-actin mRNA to normalize for initial RNA input.

Statistical analyses.
Statistical analysis was performed using Statistica 6.0 or Sigma Stat. Regression analysis has advantages over ANOVA in analytic strategies designed to take account of interlitter variability in growth, body composition, and other phenotypes as they relate to treatments applied to the various litters (24). For regression analyses, plasma-LEP and leptin mRNA levels in BAT and WAT were log transformed to normalize their respective distributions. In preliminary forward stepwise regression analyses, a best set of independent variables were selected for their ability to generate the highest predictive values (R2) for the dependent variable. Individual variables with F values <1 were excluded from the respective regression analysis. Gender was not included in the analyses as an independent variable, because F values for gender in all analyses reported here were <1.3 in primary analyses of the data. The percent of variability accounted for and the standardized correlation coefficient (ß) were used to characterize the effects of various independent variables on leptin mRNA levels in BAT and WAT and plasma-LEP.

Effects of genotype at Lepr on plasma-LEP and leptin mRNA expression in BAT and WAT in mother-reared pups were also evaluated by two-way ANOVA with litter and genotype as grouping factors to allow for the well-known litter effects on growth of suckling-age pups (28, 39, 49). Results are presented as least square means ± SE. To allow inclusion of litters containing only two of three possible genotypes at Lepr, differences between fa/fa and +/fa, and between +/fa and +/+, were examined in separate ANOVAs. In addition, ANCOVA using litter and genotype at Lepr as grouping factors and body fat mass as a covariate was performed to evaluate the effect of genotype at Lepr on plasma-LEP adjusted for body fat mass.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Effects of Leprfa gene dosage on BAT and WAT leptin mRNA levels and plasma-LEP in mother-reared rat pups.
In 10-day-old mother-reared rat pups, BAT-Lep mRNA (as a ratio to ß-actin mRNA) was 3.4-fold higher in fa/fa pups than either +/fa or +/+ pups (Fig. 1). mRNA levels for fatty acid binding protein aP2 and BAT-specific uncoupling protein Ucp1 were not significantly different among the fa/fa, +/fa, and +/+ pups (data not shown), suggesting that LEPR mediates a specific suppression of Lep gene expression in BAT. BAT-Lep mRNA was not different between the +/fa and +/+ pups (P = 0.29), which was in contrast to the Leprfa gene dose-dependent increase of WAT-Lep mRNA of the same pups (Fig. 1). In accordance with our previous observations (54), WAT-Lep mRNA in fa/fa pups was about twice that in +/fa pups, which was, in turn, about one-third higher than that in the +/+ pups. Thus homozygosity for Leprfa resulted in increased leptin mRNA levels in both BAT and WAT, whereas heterozygosity for Leprfa increases leptin mRNA levels (relative to +/+) only in WAT, suggesting that the underlying mechanisms for the LEPR-mediated suppression of leptin gene expression in WAT and BAT are different in these pups.



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Fig. 1. Least square means ±SE of leptin mRNA levels in brown adipose tissue (BAT) and white adipose tissue (WAT) (as ratio of leptin/actin) and plasma leptin concentration (plasma-LEP) after controlling for litter effects in 10-day-old mother-reared +/+ (solid bars), +/fa (hatched bars), and fa/fa (open bars) pups. Comparisons of +/+ vs. +/fa and +/fa vs. fa/fa littermates were carried out in separate groups of pups (therefore, two hatched bars for +/fa), because most litters contained only two genotypes. Statistical significance, as determined by 2-way ANOVA with litter and genotype as grouping factors, is denoted as aP < 0.001, fa/fa vs. +/fa; bP < 0.01, fa/fa vs. +/fa; cP < 0.001, +/fa vs. +/+; and dP < 0.05, +/fa vs. +/+.

 
Consistent with our earlier finding (54), plasma-LEP was also increased in a Leprfa gene dose-dependent fashion (Fig. 1), even after correcting for differences in body fat content (P = 0.023, +/+ vs. +/fa; and P < 0.001, +/fa vs. fa/fa, by ANCOVA using fat mass as a covariate). Allowing for Leprfa effects, Lep mRNA levels were 50–80% lower in BAT than in WAT of the same pups (Fig. 1). When plasma-LEP was regressed against BAT-Lep and WAT-Lep mRNA in animals for which all three parameters had been measured (R2= 0.74, n = 14, and +/fa and +/+ pups only), BAT-Lep mRNA accounted for 65% of the variation in plasma-LEP (ß = 0.70, P = 0.001). WAT-Lep mRNA, on the other hand, was only weakly correlated with plasma-LEP and accounted for only 8.5% of the variation in plasma-LEP (ß = 0.31, P = 0.09). These results suggest that BAT, despite its lower mRNA levels relative to WAT, is a major source of the circulating leptin in pups at this age, probably resulting from its significantly larger tissue mass than WAT.

Effects of experimental manipulations of sympathetic nervous activity and caloric balance on body fat content and plasma-LEP.
To identify the mechanisms for the control of leptin mRNA expression in BAT and WAT and their respective contributions to circulating plasma-LEP, variations in SNS activity, body fat content, and metabolic flux were created by experimentally manipulating ambient temperature and the rate of milk infusion in combination with NE administration during the artificial rearing of rat pups from postnatal day 4 or 8 to day 10/11. Figure 2B shows the MS-MR during the last 24 h of life of artificially reared pups. As previously reported (26), rearing pups under moderate cold loads increased MS-MR in +/fa and +/+ pups relative to pups reared under thermoneutral conditions, while MS-MR in fa/fa pups remained close to that observed under thermoneutral conditions, resulting in hypothermia in the fa/fa pups. Rearing pups of all genotypes under thermoneutral conditions equalized MS-MR at very low levels. Adding NE to the infused synthetic milk increased MS-MR to high but equal levels among fa/fa and +/fa or +/+ pups. MS-MR in NE-treated pups in the final 24 h of the experiment was about 100% above that observed in pups reared under thermoneutral conditions without NE treatment. MS-MR has been previously shown to be highly correlated with sympathetically mediated activation of BAT in pups at this age (16, 21, 22, 33) and was used as a surrogate for SNS activity in these pups. The manipulation of feeding rates in the NE-treated litters produced two groups of pups with similarly high SNS activity but markedly different body fat content and caloric balance (litters 14 and 15 vs. litter 16). These nutritional manipulations permit isolation of the effects of SNS activity from those of body fat content and/or caloric balance on leptin gene expression.

Because artificially reared pups were smaller than mother-reared pups, percentage of body fat content rather than the absolute fat mass was used to compare their adiposity to that of mother-reared animals (Fig. 2C). When pups were artificially reared under moderate cold loads (litters 8 and 9) similar to those experienced by mother-reared pups (31, 35), the Leprfa-dependent differences in body fat content and plasma-LEP were at least as pronounced as in mother-reared pups (Fig. 2, C and D), demonstrating that the artificial rearing procedure per se does not eliminate the abnormally high plasma-LEP of fa/fa pups.

Compared to cold rearing, rearing under thermoneutral conditions for 2 days (from postnatal day 8 to 10) resulted in a striking (more than 3-fold) decrease of plasma-LEP in fa/fa pups but had little effect on plasma-LEP in +/fa and +/+ pups (litters 10 and 11). This result was unexpected, because activation of the SNS has previously been shown to suppress plasma-LEP in lean mice and rats (17, 29, 48). Thus minimizing SNS activity by thermoneutral rearing had been expected to increase leptin expression in WAT in +/+ and +/fa pups instead of decreasing it in fa/fa pups. When pups were reared under thermoneutral conditions for 6 days (from postnatal day 4 to day 10/11), body fat content and plasma-LEP were indistinguishable in +/fa and fa/fa pups (litters 12 and 13). Proportional to the increase in body fat content after 6 days under thermoneutral conditions, the plasma-LEP of both fa/fa and +/fa were higher than in the pups reared under thermoneutral conditions for only 2 days. These results suggest that artificial rearing under thermoneutral conditions equalized energy expenditure between fa/fa and +/fa pups and eliminated the factor(s) that caused increased plasma-LEP in mother-reared or artificially reared fa/fa pups under moderate cold loads.

The NE treatment decreased plasma-LEP to very low, but similar, levels in both fa/fa and +/fa pups (litters 14–16), even though the body fat contents of both fa/fa and +/fa pups in litters 14 and 15 were similar to those of the fa/fa pups reared under cold load. These results demonstrated that moderate doses of NE were sufficient to suppress plasma-LEP to similarly low levels in both fa/fa and +/fa pups without concomitantly decreasing body fat content.

Effects of experimental manipulations of SNS activity and caloric balance on leptin expression in WAT and BAT.
We examined leptin mRNA levels in WAT and BAT of pups in which caloric balance and SNS activity had been manipulated to introduce variations in metabolic flux and/or energy storage that were independent of altered SNS activity. In the pups artificially reared under moderate cold loads, WAT-Lep mRNA showed a similar Leprfa dose-dependent relationship among fa/fa, +/fa and +/+ pups as was seen in mother-reared pups, although the sample size for this group of pups was too small to yield statistically significant differences between the genotypes (Fig. 3A). Minimizing SNS activity by thermoneutral rearing for either 2 or 6 days equalized WAT-Lep mRNA in the +/fa and fa/fa pups by decreasing Lep expression in fa/fa pups. Lower SNS activity would be expected to increase Lep expression in pups of all genotypes (13, 48). The unexpected result here suggests that SNS activity per se is not an important determinant of Lep expression in WAT at this age (see below). Increasing SNS activity by NE treatment was not sufficient to decrease or equalize WAT-Lep mRNA in fa/fa and +/fa pups from litters with high body fat content (FatHI, Figs. 2 and 3). WAT-Lep mRNA was nearly twofold higher in the fa/fa than the +/fa pups (P = 0.055), despite their similar body fat contents (Fig. 2C). In comparison, decreasing body fat content and caloric balance by reducing feeding rate in the NE-treated litter (FatLO) markedly suppressed WAT-Lep mRNA levels in both fa/fa and +/fa pups, implying that energy flux and/or fat content play an important role in regulating leptin expression in WAT of these pups.

The effects of rearing conditions on leptin gene expression in BAT of the same artificially reared animals were very different from those in WAT observed above. BAT-Lep mRNA in cold-reared pups was much lower than that in mother-reared pups (Fig. 3B). But, as in mother-reared pups, BAT-Lep mRNA in fa/fa pups was severalfold higher than that of their +/+ and +/fa littermates, whose levels were not different from each other. Thermoneutral rearing pups for 2 or 6 days increased BAT-Lep mRNA in both +/fa and fa/fa pups in a time-dependent manner to much higher levels, relative to pups either mother-reared or artificially reared under moderate cold loads. Thermoneutral rearing also completely eliminated the differences in BAT-Lep mRNA between +/fa and fa/fa pups. Thus, in contrast to WAT-Lep mRNA, BAT-Lep mRNA showed the expected increase when SNS activity was decreased by rearing under thermoneutral conditions. The potent impact of SNS activity on BAT-Lep mRNA is also emphasized by the dramatic suppression and equalization of BAT-Lep mRNA to very low levels in NE-treated +/fa and fa/fa pups, even in litters in which increased rate of milk infusion had prevented a decrease of body fat content (FatHI, Figs. 2 and 3).

To further characterize the effects of SNS activity, metabolic flux, and/or energy storage and genotype at Lepr on leptin mRNA expression in adipose tissues, we performed multiple regression analyses using BAT-Lep mRNA and WAT-Lep mRNA as dependent variables, and MR (as a surrogate for SNS activity), caloric balance and body fat mass (as surrogates for metabolic flux and energy storage), FFDM, and genotype at Lepr locus as independent variables. FFDM, which is a more precise measure of lean body mass than wet body mass, was included in these analyses to adjust for differences in the metabolic size among the pups. BAT-Lep mRNA showed a strong inverse correlation with MR (ß = -0.78, P < 0.001) and a moderate positive correlation with body fat mass (ß = 0.37, P < 0.001) (total R2 = 0.93) (Table 1). In stepwise regression analysis, MR and fat mass accounted for 87% and 5% of the variation in BAT-Lep mRNA, respectively. WAT-Lep mRNA, in contrast, was mainly influenced by caloric balance (ß = 0.55, P < 0.001) and fat mass (ß = 0.39, P < 0.01)(total R2 = 0.53) (Table 1). In stepwise regression analysis, caloric balance and fat mass accounted for 40% and 6% of the variation of WAT-Lep mRNA, respectively. MR also had a weak positive impact on WAT-Lep mRNA, accounting for 5% of the variation (ß = 0.36, P < 0.01), in contradistinction to the negative impact seen in BAT-Lep and in previous studies of WAT (17, 29, 48). This unexpected result could be attributed to the very low WAT-Lep mRNA in fa/fa pups reared under thermoneutral conditions without NE treatment. Indeed, excluding these pups in the regression analysis eliminated the correlation of MR with WAT-Lep mRNA.


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Table 1. Multiple regression analysis of effects of MR, caloric balance, fat mass, FFDM, and genotype at Lepr on plasma-LEP and leptin mRNA expression in BAT and WAT and of the relative contributions of leptin production in BAT and WAT to plasma-LEP

 
The dominant effect of SNS activity on BAT-Lep mRNA and its relative lack of importance with regard to WAT-Lep mRNA in pups at this age were also confirmed by simple correlation analyses. Although WAT-Lep mRNA was not significantly correlated with either MR (r = -0.19) or MS-MR (r = -0.08), the correlation between BAT-Lep mRNA and either MR (r = -0.93) or MS-MR (r = -0.92) was remarkably high (P < 0.001).

Surprisingly, copy number of Leprfa had no significant effect on either BAT-Lep mRNA or WAT-Lep mRNA when MR, body fat mass, and caloric balance were considered in the regression analysis, suggesting that the Leprfa may not have a direct (autocrine) effect on leptin mRNA expression in adipose tissues of 10-day-old rat pups. Thus the observed effects of Lepr-mediated feedback suppression on leptin mRNA expression in mother-reared pups appear to be due mainly to LEPR-mediated effects on SNS activity in BAT and to effects on energy flux and/or energy storage in WAT.

The role of WAT and BAT leptin mRNA levels and associated physiological parameters in determining plasma-LEP.
To assess the relative contributions of rates of leptin expression in BAT and WAT to the circulating leptin concentrations in artificially reared pups, we performed multiple regression analyses with plasma-LEP as the dependent variable and BAT-Lep and WAT-Lep mRNA levels and genotype at Lepr as independent variables (Table 1). It was noted that all of the fa/fa pups reared under cold loads, and 2 of the 34 fa/fa pups reared under thermoneutral conditions, had plasma-LEP (>10 ng/ml) much higher than the other artificially reared pups. Multiple regression analysis with all artificially reared pups gave a lower total R2 = 0.61 than in the analysis when pups with plasma-LEP > 10 ng/ml were excluded (R2 = 0.70). Under the latter analysis paradigm, BAT-Lep mRNA accounts for 60% of the variation in plasma-LEP, whereas WAT-Lep mRNA accounts for only ~3% of the variation, consistent with the results obtained in mother-reared pups.

Genotype at Lepr accounted for 7% of the variation in plasma-LEP, suggesting that the functional integrity of LEPR has effects on plasma-LEP by mechanisms separate from its effects on leptin mRNA expression in BAT and WAT. Inclusion of MR (surrogate for SNS activity) as an additional independent variable in the regression analysis did not increase the total R2 value (data not shown). Because of the significant effect on Lep expression of genotype at Lepr, we also evaluated +/fa and fa/fa pups in separate regression analyses. The correlation of BAT-Lep mRNA or WAT-Lep mRNA with plasma-LEP was higher in fa/fa pups (ß = 0.84 and 0.31, respectively) than in +/fa pups (ß = 0.73 and 0.13, respectively). However, the analyses confirmed the predominant effects of BAT, relative to WAT, on plasma-LEP in both fa/fa and +/fa pups.

To directly assess the relationship between leptin mRNA expression in adipose tissues and plasma-LEP in pups under the various rearing conditions, we performed simple correlation analysis of WAT-Lep mRNA or BAT-Lep mRNA with plasma-LEP (Fig. 4, A and B). These analyses support the conclusions based upon the multiple regression analysis described above: plasma-LEP values were very strongly correlated with BAT-Lep mRNA (r = 0.78, P < 0.001) (Fig. 4B) but less strongly with WAT-Lep mRNA (r = 0.39, P < 0.01) (Fig. 4A). The least square means of plasma-LEP, WAT-Lep mRNA, and BAT-Lep mRNA from mother-reared pups (after controlling for litter effects, oversized solid symbols in Fig. 4) are included to permit a direct comparison between mother-reared and artificially reared pups. The means for mother-reared +/+ and +/fa pups were within the range of artificially reared +/+ and +/fa pups. However, the mean plasma-LEP of the mother-reared fa/fa pups was above 10 ng/ml, similar to those found in the fa/fa pups artificially reared under cold loads (large solid squares with dot and cross, in Fig. 4). In contrast, only 2 of 34 fa/fa pups reared under thermoneutral conditions had plasma-LEP >10 ng/ml (open square with cross, Fig. 4). Thus it appears that cold exposure increased plasma-LEP in both mother-reared and artificially reared fa/fa pups. But such an effect was not observed in the lean (+/fa and +/+) pups.

We also analyzed MR, caloric balance, body fat content, and genotype at Lepr for their effects on plasma-LEP in artificially reared pups by multiple regression analysis (Table 1). As expected, plasma-LEP was inversely related to MR (ß = -0.58, P < 0.001) and positively influenced by body fat content (ß = 0.50, P < 0.001). Caloric balance and FFDM also showed a weak, negative correlation with plasma-LEP. Total R2 was 0.70. No additional effect of genotype at Lepr on plasma-LEP was detected, suggesting that the effect of the Leprfa gene on plasma-LEP observed in mother-reared pups was mediated mostly through the effects of Leprfa gene on SNS activity, metabolic flux, and/or energy storage.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Differential regulation of leptin mRNA expression in BAT and WAT.
These data indicate that leptin mRNA expression is differentially regulated in BAT and WAT of 10-day-old rat pups. BAT-Lep mRNA levels were higher in pups artificially reared under thermoneutral conditions, which decreases SNS activity, and were lower in pups treated with NE, which mimics increased SNS activity. This strong, negative correlation of BAT-Lep with SNS activity in the artificially reared pups regardless of their genotype at Lepr suggests that SNS activity plays a major role in regulating leptin expression in BAT. In contrast, the lack of significant, negative correlation between WAT-Lep mRNA and SNS activity suggests that SNS activity plays a minimal, if any, role in regulating leptin expression in WAT. On the other hand, the significant positive correlations of WAT-Lep mRNA with caloric balance and body fat mass support earlier observations that metabolic flux and/or energy storage play an important role in regulating leptin mRNA expression in WAT (51, 52).

The failure of exogenous NE to suppress Lep expression in WAT of the pups in NE-treated FatHI group (Fig. 3) was surprising. Previous reports have indicated that cold stress or administration of catecholamines or ß3-adrenergic receptor agonists suppresses leptin gene expression in WAT of adult lean mice, although the effects of these manipulations on leptin expression in WAT were much less potent than in BAT (29, 48). Since most reported effects of SNS activation or administration of ß-adrenergic agonist on leptin gene expression in adult animals were obtained by experiments without appropriate control of substrate flux in adipocytes (13, 17, 29, 48), it is possible that the sympathetic-mediated effects on leptin gene expression are secondary to sympathetic-mediated lipolysis in adipocytes. This idea is consistent with the fact that the suppression of leptin mRNA levels in adipocytes by catecholamines appears to be coupled to the synthesis of cAMP, which also stimulates lipolysis (13, 23, 43). Changes in fatty acids flux due to SNS-mediated lipolysis in adipocytes may influence leptin gene expression by effects on the hexosamine biosynthetic pathway (51, 52) or by other mechanisms. Consistent with our results in suckling pups, Moinat et al. (29) have also shown that in adult rats, food restriction decreases leptin mRNA levels in WAT to a much greater extent than either cold-stimulation or administration of ß3-adrenergic agonist. Based on these results, Moinat et al. (29) suggested that leptin gene expression in WAT might be more closely associated with alterations in cell lipid content and/or substrate flux than with changes in SNS activity per se. Thus our results in suckling pups agree with those of Moinat et al. (29) in adult rats with regard to the relatively higher sensitivity of leptin gene expression in WAT to changes in substrate flux than to changes in SNS activity per se, even though the relative size of BAT and WAT and their contributions to the circulating leptin concentration differ significantly during the suckling period and adult life. However, it is important to note that our results do not exclude the possibility of regulation of leptin gene expression in WAT by SNS activity in either pups or adult rats, but only emphasize the relative importance of substrate flux in control of leptin gene expression in WAT. The more potent effects of metabolic flux into adipocytes/energy storage may mask the relatively weak effects of enhanced SNS activity on Lep expression in WAT of pups in NE-treated FatHI group (Fig. 3). It is also worth noting that the total R2 for SNS activity, body fat mass, and caloric balance in predicting WAT Lep mRNA levels was relative low (0.53), compared with 0.93 in BAT, suggesting that factors other than SNS activity or metabolic flux/energy storage may be involved in mediating the regulation of WAT-Lep expression in the pups.

Contributions of leptin expression in BAT and WAT to plasma-LEP.
The higher degree of correlation between plasma-LEP and leptin mRNA expression in BAT than in WAT in both mother-reared and artificially reared pups suggests that BAT is a more significant source of circulating leptin than WAT in 10-day-old rat pups. Since transcription rates of leptin (relative to actin) were severalfold lower in BAT than WAT in mother-reared and artificially reared pups under cold load or NE treatment, the greater mass of BAT (vs. WAT) at this age may partly account for the higher total leptin production by BAT than WAT. Although the relative contribution of WAT to circulating leptin levels, compared with that of BAT, is apparently lower in these 10-day-old pups, the contribution of the WAT depot increases with age as WAT mass increases. In adult animals, plasma-LEP correlates highly with both WAT-Lep mRNA and the mass of WAT (18, 25, 37).

Interestingly, although BAT-Lep mRNA expression rate was more closely correlated with plasma-LEP, plasma-LEP was not always correlated with BAT-Lep mRNA under all artificial rearing conditions. For example, the dramatic increase of BAT-Lep mRNA in both fa/fa and +/fa pups reared under thermoneutral conditions without NE for 6 days was not accompanied by high plasma-LEP. On the other hand, the very high plasma-LEP (>10 µg/ml) levels that were observed in fa/fa pups artificially reared under cold loads were associated with WAT-Lep mRNA and BAT-Lep mRNA that were either similar or lower than those seen in pups reared under thermoneutral condition without NE treatment for 6 days. These results suggest that plasma-LEP in these pups may also be regulated by mechanisms that are not associated with leptin production in BAT and WAT, such as the clearance of the circulating leptin. The kidney has been shown to be a major site for clearance of circulating leptin by glomerular filtration (11, 50). Hypothermia decreases renal blood flow and glomerular filtration rate by non-SNS-mediated mechanisms (4, 6). Thus decreased clearance of circulating leptin may contribute to the marked increase of plasma-LEP in hypothermic relative to euthermic fa/fa pups.

Mechanisms of Lepr-mediated feedback suppression of leptin gene expression.
The importance of leptin signaling in energy homeostasis during the early postnatal life of rat pups is clearly demonstrated by the onset of increased fat deposition in pups that are either heterozygous or homozygous for the mutant leptin receptor (Leprfa) (28, 39, 49, 54). Leptin administration also alters the circadian rhythm of oxygen consumption and reduces time spent in a torpid state in suckling pups (44). The Leprfa gene dose-dependent increases in BAT and WAT Lep mRNA levels, as well as plasma-LEP, suggest that leptin signaling plays a role, directly or indirectly, in controlling leptin expression at this age. The increased leptin mRNA in BAT and WAT of fa/fa pups was not associated with a general increase of gene transcription rates, since BAT Ucp1 and aP2 mRNA levels were not significantly different between mother-reared fa/fa and +/fa pups (data not shown). The lack of effect of Leprfa on Ucp1 mRNA levels in the BAT of the 10-day-old fa/fa pups is consistent with a previous report in which decreased thermoregulatory thermogenesis in 10-day-old fa/fa rats was shown to be due to decreased GDP binding capacity of UCP1 protein, rather than to decreased Ucp1 mRNA or protein levels (1). However, significant decreases in BAT Ucp1 mRNA and protein levels have been found in older fa/fa rats relative to lean littermates (1).

The finding that BAT-Lep mRNA levels were not different between fa/fa and +/fa pups when their SNS activity was equalized by thermoneutral rearing suggests that SNS constitutes the major mechanism for the LEPR-mediated feedback suppression on leptin gene expression in BAT of pups at this age. The apparent insensitivity of WAT leptin mRNA levels to NE treatment in both fa/fa and +/fa pups suggests that decreased SNS activity is not the primary mechanism for the increased WAT leptin mRNA levels in fa/fa mother-reared pups. The marked difference of WAT-Lep mRNA between NE-treated pups with high (FatHI) and low (FatLO) body fat content and caloric balance emphasizes the importance of metabolic flux/energy storage in regulating leptin expression in WAT (Fig. 3).

Thermoneutral rearing equalized WAT-Lep mRNA in fa/fa and +/fa pups by decreasing WAT leptin expression in fa/fa pups, rather than increasing WAT-Lep mRNA in +/fa pups, as one would have predicted based on the effects of thermoneutral rearing on BAT-Lep mRNA and known effects of SNS activity on leptin mRNA expression in WAT of adult animals (17, 29, 48). One possible factor responsible for the higher WAT-Lep mRNA levels in fa/fa than +/fa pups reared in a colder ambient temperature (either mother-reared or artificially reared) may be the general physiological stress induced by hypothermia in fa/fa pups. Rearing pups under thermoneutral conditions prevents hypothermia and the increase of WAT-Lep mRNA in fa/fa. Glucocorticoids, which increase leptin expression in adipocytes, might mediate the increased leptin expression in WAT of fa/fa pups reared under relatively cold conditions (43). Experimental manipulation of glucocorticoid status would address this possibility. Alternatively, the relative increases of metabolic flux toward WAT due to the decreased energy expenditure for thermogenesis in BAT of fa/fa pups may be responsible for the higher WAT-Lep mRNA in fa/fa than +/fa pups under cold loads.

No significant effect of genotype at Lepr on leptin mRNA levels in BAT and WAT was detected in artificially reared pups after controlling for SNS activity, caloric balance, and body fat content. This result suggests that the effects of Leprfa gene dosage on leptin mRNA levels in BAT and WAT observed in mother-reared pups are largely mediated by Leprfa effects on SNS activity and metabolic flux/energy storage. The effects of Lepr on SNS and metabolic flux/energy storage may be mediated mainly by LEPR in the hypothalamus (5, 47). However, leptin’s effects on metabolic flux in WAT and BAT conveyed directly by leptin receptors on adipocytes may also be involved in the feedback regulation of leptin on its own gene expression (27, 40, 42, 56).

In summary, the results reported here show: 1) Leptin mRNA expressions in BAT and WAT of 10-day-old rat pups are differently regulated; BAT-Lep mRNA is strongly suppressed by SNS activity, whereas WAT-Lep mRNA is primarily influenced by adipocyte metabolic flux/energy storage. 2) Although BAT-Lep mRNA levels are severalfold lower than WAT-Lep mRNA levels, plasma-LEP levels in pups of this age are predominantly related to BAT-Lep mRNA expression rates. 3) Leptin expression in both BAT and WAT is increased in fa/fa pups compared with +/fa and +/+ pups. Decreased SNS activity is mainly responsible for the increased leptin mRNA level in BAT in fa/fa pups, but increased metabolic flux/energy storage is at least partly responsible for the elevation of leptin mRNA level in WAT of fa/fa pups at this age.


    ACKNOWLEDGMENTS
 
This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52431 and DK-26687 (to R. L. Leibel) and by Deutsche Forschungsgemeinschaft Schm 680/2 (to I. Schmidt). Y. Zhang is the recipient of a Career Development Award from the American Diabetes Association.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: Y. Zhang, Division of Molecular Genetics, Columbia Univ., Russ Berrie Pavilion, 1150 St. Nicholas Ave., New York, NY 10032 (E-mail: yz84{at}columbia.edu).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ashwell M, Holt S, Jennings G, Stirling DM, Trayhurn P, and York DA. Measurement by radioimmunoassay of the mitochondrial uncoupling protein from brown adipose tissue of obese (ob/ob) mice and Zucker (fa/fa) rats at different ages. FEBS Lett 179: 233–237, 1985.[ISI][Medline]
  2. Bamshad M, Song CK, and Bartness TJ. CNS origins of the sympathetic nervous system outflow to brown adipose tissue. Am J Physiol Regulatory Integrative Comp Physiol 276: R1569–R1578, 1999.[Abstract/Free Full Text]
  3. Bartness TJ and Bamshad M. Innervation of mammalian white adipose tissue: implications for the regulation of total body fat. Am J Physiol Regulatory Integrative Comp Physiol 275: R1399–R411, 1998.[Abstract/Free Full Text]
  4. Broman M and Kallskog O. The effects of hypothermia on renal function and haemodynamics in the rat. Acta Physiol Scand 153: 179–184, 1995.[ISI][Medline]
  5. Campfield LA, Smith FJ, Guisez Y, Devos R, and Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269: 546–549, 1995.[ISI][Medline]
  6. Chapman BJ, Withey WR, and Munday KA. Autoregulation of renal blood flow in dogs at normal body temperature and at 27 degrees C. Clin Sci Mol Med Suppl 48: 501–508, 1975.[Medline]
  7. Chua, SC Jr, White DW, Wu-Peng XS, Liu SM, Okada N, Kershaw EE, Chung WK, Power-Kehoe L, Chua M, Tartaglia LA, and Leibel RL. Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr). Diabetes 45: 1141–1143, 1996.[Abstract]
  8. Chung WK, Belfi K, Chua M, Wiley J, Mackintosh R, Nicolson M, Boozer CN, and Leibel RL. Heterozygosity for Lep(ob) or Lepr(db) affects body composition and leptin homeostasis in adult mice. Am J Physiol Regulatory Integrative Comp Physiol 274: R985–R990, 1998.[Abstract/Free Full Text]
  9. Cinti S, Frederich RC, Zingaretti MC, De Matteis R, Flier JS, and Lowell BB. Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue. Endocrinology 138: 797–804, 1997.[Abstract/Free Full Text]
  10. Coleman DL. Thermogenesis in diabetes-obesity syndromes in mutant mice. Diabetologia 22: 205–111, 1982.[ISI][Medline]
  11. Cumin F, Baum HP, and Levens N. Mechanism of leptin removal from the circulation by the kidney. J Endocrinol 155: 577–585, 1997.[Abstract/Free Full Text]
  12. Cusin I, Rohner-Jeanrenaud F, Stricker-Krongrad A, and Jeanrenaud B. The weight-reducing effect of an intracerebroventricular bolus injection of leptin in genetically obese fa/fa rats. Reduced sensitivity compared with lean animals. Diabetes 45: 1446–1450, 1996.[Abstract]
  13. Deng C, Moinat M, Curtis L, Nadakal A, Preitner F, Boss O, Assimacopoulos-Jeannet F, Seydoux J, and Giacobino JP. Effects of beta-adrenoceptor subtype stimulation on obese gene messenger ribonucleic acid and on leptin secretion in mouse brown adipocytes differentiated in culture. Endocrinology 138: 548–552, 1997.[Abstract/Free Full Text]
  14. Dessolin S, Schalling M, Champigny O, Lonnqvist F, Ailhaud G, Dani C, and Ricquier D. Leptin gene is expressed in rat brown adipose tissue at birth. FASEB J 11: 382–387, 1997.[Abstract/Free Full Text]
  15. Devaskar SU, Ollesch C, Rajakumar RA, and Rajakumar PA. Developmental changes in ob gene expression and circulating leptin peptide concentrations. Biochem Biophys Res Commun 238: 44–47, 1997.[ISI][Medline]
  16. Doring H, Kortner G, Meyer K, and Schmidt I. Do changes of sympathetically stimulated thermogenesis underlie the developmental changes in the cold defense of rat pups? In: Integrative and Cellular Aspects of Autonomic Functions: Temperature and Osmoregulation, edited by Pleschka K and Gerstberger R. London: Libbey, 1994, p. 285–294.
  17. Evans BA, Agar L, and Summers RJ. The role of the sympathetic nervous system in the regulation of leptin synthesis in C57BL/6 mice. FEBS Lett 444: 149–154, 1999.[ISI][Medline]
  18. Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, and Flier JS. Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med 1: 1311–1314, 1995.[ISI][Medline]
  19. Fruhbeck G, Aguado M, and Martinez JA. In vitro lipolytic effect of leptin on mouse adipocytes: evidence for a possible autocrine/paracrine role of leptin. Biochem Biophys Res Commun 240: 590–594, 1997.[ISI][Medline]
  20. Kaul R, Heldmaier G, and Schmidt I. Defective thermoregulatory thermogenesis does not cause onset of obesity in Zucker rats. Am J Physiol Endocrinol Metab 259: E11–E18, 1990.[Abstract/Free Full Text]
  21. Kortner G, Petrova O, Vogt S, and Schmidt I. Sympathetically and nonsympathetically mediated onset of excess fat deposition in Zucker rats. Am J Physiol Endocrinol Metab 267: E947–E953, 1994.[Abstract/Free Full Text]
  22. Kortner G, Schildhauer K, Petrova O, and Schmidt I. Rapid changes in metabolic cold defense and GDP binding to brown adipose tissue mitochondria of rat pups. Am J Physiol Regulatory Integrative Comp Physiol 264: R1017–R1023, 1993.[Abstract/Free Full Text]
  23. Kosaki A, Yamada K, and Kuzuya H. Reduced expression of the leptin gene (ob) by catecholamine through a G(S) protein-coupled pathway in 3T3-L1 adipocytes. Diabetes 45: 1744–1749, 1996.[Abstract]
  24. Kraeft S, Schwarzer K, Eiden S, Nuesslein-Hildesheim B, Preibisch G, and Schmidt I. Leptin responsiveness and gene dosage for leptin receptor mutation (fa) in newborn rats. Am J Physiol Endocrinol Metab 276: E836–E842, 1999.[Abstract/Free Full Text]
  25. Maffei M, Fei H, Lee GH, Dani C, Leroy P, Zhang Y, Proenca R, Negrel R, Ailhaud G, and Friedman JM. Increased expression in adipocytes of ob RNA in mice with lesions of the hypothalamus and with mutations at the db locus. Proc Natl Acad Sci USA 92: 6957–6960, 1995.[Abstract]
  26. Markewicz B, Kuhmichel G, and Schmidt I. Onset of excess fat deposition in Zucker rats with and without decreased thermogenesis. Am J Physiol Endocrinol Metab 265: E478–E486, 1993.[Abstract/Free Full Text]
  27. May JM. Rat adipocyte utilization of different substrates: effects of cell size and the control of lipogenesis. Lipids 17: 626–633, 1982.[ISI][Medline]
  28. Meierfrankenfeld B, Abelenda M, Jauker H, Klingenspor M, Kershaw EE, Chua SC Jr, Leibel RL, and Schmidt I. Perinatal energy stores and excessive fat deposition in genetically obese (fa/fa) rats. Am J Physiol Endocrinol Metab 270: E700–E708, 1996.[Abstract/Free Full Text]
  29. Moinat M, Deng C, Muzzin P, Assimacopoulos-Jeannet F, Seydoux J, Dulloo AG, and Giacobino JP. Modulation of obese gene expression in rat brown and white adipose tissues. FEBS Lett 373: 131–134, 1995.[ISI][Medline]
  30. Nuesslein B, Petrova O, Schildhauer K, and Schmidt I. Morning depression of cold defense in juvenile rats. In: Integrative and Cellular Aspects of Autonomic Functions: Temperature and Osmoregulation, edited by Pleschka K and Gerstberger R. London: Libbey, 1994, p. 285–294.
  31. Nuesslein B and Schmidt I. Development of circadian cycle of core temperature in juvenile rats. Am J Physiol Regulatory Integrative Comp Physiol 259: R270–R276, 1990.[Abstract/Free Full Text]
  32. Planche E, Joliff M, de Gasquet P, and Leliepvre X. Evidence of a defect in energy expenditure in 7-day-old Zucker rat (fa/fa). Am J Physiol Endocrinol Metab 245: E107–E113, 1983.[Abstract/Free Full Text]
  33. Redlin U, Nuesslein B, and Schmidt I. Circadian changes of brown adipose tissue thermogenesis in juvenile rats. Am J Physiol Regulatory Integrative Comp Physiol 262: R504–R508, 1992.[Abstract/Free Full Text]
  34. Rosenbaum M, Nicolson M, Hirsch J, Heymsfield SB, Gallagher D, Chu F, and Leibel RL. Effects of gender, body composition, and menopause on plasma concentrations of leptin. J Clin Endocrinol Metab 81: 3424–3427, 1996.[Abstract]
  35. Schmidt I. The role of juvenile thermoregulatory thermogenesis in the development of normal energy balance or obesity. In: Thermotherapy for Neoplasia, Inflammation and Pain, edited by Kosaka M, Sugahara T, Schmidt KL, and Simon E. Tokyo: Springer Verlag, 2000.
  36. Schmidt I, Kaul R, and Heldmaier G. Thermoregulation and diurnal rhythms in 1-week-old rat pups. Can J Physiol Pharmacol 65: 1355–1364, 1987.[ISI][Medline]
  37. Schwartz MW, Peskind E, Raskind M, Boyko EJ, and Porte D Jr. Cerebrospinal fluid leptin levels: relationship to plasma levels and to adiposity in humans. Nat Med 2: 589–593, 1996.[ISI][Medline]
  38. Schwartz MW, Seeley RJ, Campfield LA, Burn P, and Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98: 1101–1106, 1996.[Abstract/Free Full Text]
  39. Schwarzer K, Doring H, and Schmidt I. Different physiological traits underlying increased body fat of fatty (fa/fa) and heterozygous (+/fa) rats. Am J Physiol Endocrinol Metab 272: E100–E106, 1997.[Abstract/Free Full Text]
  40. Shimabukuro M, Koyama K, Chen G, Wang MY, Trieu F, Lee Y, Newgard CB, and Unger RH. Direct antidiabetic effect of leptin through triglyceride depletion of tissues. Proc Natl Acad Sci USA 94: 4637–4641, 1997.[Abstract/Free Full Text]
  41. Shimazu T. Central nervous system regulation of liver and adipose tissue metabolism. Diabetologia 20: 343–356, 1981.[ISI][Medline]
  42. Siegrist-Kaiser CA, Pauli V, Juge-Aubry CE, Boss O, Pernin A, Chin WW, Cusin I, Rohner-Jeanrenaud F, Burger AG, Zapf J, and Meier CA. Direct effects of leptin on brown and white adipose tissue. J Clin Invest 100: 2858–2864, 1997.[Abstract/Free Full Text]
  43. Slieker LJ, Sloop KW, Surface PL, Kriauciunas A, LaQuier F, Manetta J, Bue-Valleskey J, and Stephens TW. Regulation of expression of ob mRNA and protein by glucocorticoids and cAMP. J Biol Chem 271: 5301–5304, 1996.[Abstract/Free Full Text]
  44. Stehling O, Doring H, Ertl J, Preibisch G, and Schmidt I. Leptin reduces juvenile fat stores by altering the circadian cycle of energy expenditure. Am J Physiol Regulatory Integrative Comp Physiol 271: R1770–1774, 1996.[Abstract/Free Full Text]
  45. Stehling O, Doring H, Nuesslein-Hildesheim B, Olbort M, and Schmidt I. Leptin does not reduce body fat content but augments cold defense abilities in thermoneutrally reared rat pups. Pflügers Arch 434: 694–697, 1997.
  46. Tai TAC, Jennermann C, Brown KK, Oliver BB, MacGinnitie MA, Wilkison WO, Brown HR, Lehmann JM, Kliewer SA, Morris DC, and Graves RA. Activation of the nuclear receptor peroxisome proliferator-activated receptor gamma promotes brown adipocyte differentiation. J Biol Chem 271: 29909–29914, 1996.[Abstract/Free Full Text]
  47. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muri C, Sanker S, Moriarty A, Moore K, Smutko JS, Mays GG, Woolf EA, Monroe CA, and Tepper RI. Identification and expression cloning of a leptin receptor, OB-R. Cell 83: 1263–1271, 1995.[ISI][Medline]
  48. Trayhurn P, Duncan JS, Rayner DV, and Hardie LJ. Rapid inhibition of ob gene expression and circulating leptin levels in lean mice by the beta 3-adrenoceptor agonists BRL 35135A and ZD2079. Biochem Biophys Res Commun 228: 605–610, 1996.
  49. Truett GE, Tempelman RJ, and Walker JA. Codominant effects of the fatty (fa) gene during early development of obesity. Am J Physiol Endocrinol Metab 268: E15–E20, 1995.[Abstract/Free Full Text]
  50. Van Heek M, Mullins DE, Wirth MA, Graziano MP, Fawzi AB, Compton DS, France CF, Hoos LM, Casale RL, Sybertz EJ, Strader CD, and Davis HR Jr. The relationship of tissue localization, distribution and turnover to feeding after intraperitoneal 125I-leptin administration to ob/ob and db/db mice. Horm Metab Res 28: 653–658, 1996.[ISI][Medline]
  51. Wang J, Liu R, Hawkins M, Barzilai N, and Rossetti L. A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature 393: 684–688, 1998.[ISI][Medline]
  52. Wang J, Liu R, Liu L, Chowdhury R, Barzilai N, Tan J, and Rossetti L. The effect of leptin on Lep expression is tissue-specific and nutritionally regulated. Nat Med 5: 895–899, 1999.[ISI][Medline]
  53. Young JB and Landsberg L. Diminished sympathetic nervous system activity in genetically obese (ob/ob) mouse. Am J Physiol Endocrinol Metab 245: E148–E154, 1983.[Abstract/Free Full Text]
  54. Zhang Y, Olbort M, Schwarzer K, Nuesslein-Hildesheim B, Nicolson M, Murphy E, Kowalski TJ, Schmidt I, and Leibel RL. The leptin receptor mediates apparent autocrine regulation of leptin gene expression. Biochem Biophys Res Commun 240: 492–495, 1997.[ISI][Medline]
  55. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, and Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425–432, 1994.[ISI][Medline]
  56. Zhou YT, Shimabukuro M, Koyama K, Lee Y, Wang MY, Trieu F, Newgard CB, and Unger RH. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation. Proc Natl Acad Sci USA 94: 6386–6390, 1997.[Abstract/Free Full Text]