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Endocrinology, doi:10.1210/en.2007-0261
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Endocrinology Vol. 148, No. 8 3987-3997
Copyright © 2007 by The Endocrine Society

Disruption of Peripheral Leptin Signaling in Mice Results in Hyperleptinemia without Associated Metabolic Abnormalities

Kaiying Guo, Julie E. McMinn, Thomas Ludwig, Yi-Hao Yu, Guoqing Yang, Lulu Chen, Daniella Loh, Cai Li, Streamson Chua, Jr. and Yiying Zhang

Division of Molecular Genetics (K.G., J.E.M., L.C., D.L., S.C., Y.Z.), Department of Pediatrics, Naomi Berrie Diabetes Center (Y.Z.) and Departments of Cell Biology and Anatomy (T.L.) and Medicine (Y.-H.Y.), Columbia University, College of Physicians and Surgeons, New York, New York 10032; and Department of Physiology (G.Y., C.L.), The University of Texas Southwestern Medical Center, Dallas, Texas 75390

Address all correspondence and requests for reprints to: Yiying Zhang, Ph.D., Division of Molecular Genetics, Columbia University, Russ Berrie Pavilion, 1150 St. Nicholas Avenue, New York, New York 10032. E-mail: yz84{at}columbia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although central leptin signaling appears to play a major role in the regulation of food intake and energy metabolism, the physiological role of peripheral leptin signaling and its relative contribution to whole-body energy metabolism remain unclear. To address this question, we created a mouse model (Cre-Tam mice) with an intact leptin receptor in the brain but a near-complete deletion of the signaling domain of leptin receptor in liver, adipose tissue, and small intestine using a tamoxifen (Tam)-inducible Cre-LoxP system. Cre-Tam mice developed marked hyperleptinemia (~4-fold; P < 0.01) associated with 2.3-fold increase (P < 0.05) in posttranscriptional production of leptin. Whereas this is consistent with the disruption of a negative feedback regulation of leptin production in adipose tissue, there were no discernable changes in energy balance, thermoregulation, and insulin sensitivity. Hypothalamic levels of phosphorylated signal transducer and activator of transcription 3, neuropeptide expression, and food intake were not changed despite hyperleptinemia. The percentage of plasma-bound leptin was markedly increased (90.1–96 vs. 41.8–74.7%; P < 0.05), but plasma-free leptin concentrations remained unaltered in Cre-Tam mice. We conclude from these results that 1) the relative contribution to whole-body energy metabolism from peripheral leptin signaling is insignificant in vivo, 2) leptin signaling in adipocyte constitutes a distinct short-loop negative feedback regulation of leptin production that is independent of tissue metabolic status, and 3) perturbation of peripheral leptin signaling alone, although increasing leptin production, may not be sufficient to alter the effective plasma levels of leptin because of the counter-regulatory increase in the level of leptin binding protein(s).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN IS PRODUCED predominantly in adipocytes and plays a pivotal role in regulating food intake, energy expenditure, and glucose homeostasis (1, 2, 3, 4). The effects of leptin on energy balance and metabolism are mediated by its cognate receptor (LEPR), which is a member of the class I cytokine receptor superfamily (5). Several splicing variants of LEPR, including the membrane-bound isoforms (Ra to Rd) and a soluble isoform (Re), have been identified (5, 6, 7). Rb, the only isoform capable of signaling via the Janus kinases and signal transducer and activator of transcription (STAT) pathway, is responsible for mediating the metabolic effects of leptin (6, 8). LEPR is expressed in many tissues, and the Rb isoform is expressed at high levels in the hypothalamus and other regions of the brain and at substantially lower levels in many peripheral tissues, including adipose tissues, skeletal muscle, adrenal glands, pancreatic islets, liver, kidney, lymph nodes, and gonads (5, 6, 8).

LEPR in the central nervous system (CNS), through a complex neuroendocrine network and autonomic nervous system that are yet to be fully delineated, appears to play a dominant role over that in peripheral tissues in mediating the effects of the hormone on food intake and energy metabolism (2, 9, 10, 11, 12, 13, 14). In an attempt to assess the relative role of leptin signaling in the brain vs. that in the peripheral tissues, Cohen et al. (12) showed that neuron-specific deletion of Lepr (OBRSynIKO) causes obesity, whereas liver-specific deletion of Lepr has no discernable effect on energy metabolism. Although adiposity in OBRSynIKO mice correlates inversely with the level of residual LEPR expression in the CNS, the obese phenotype is significantly milder (12) than that of the global Lepr-deficient mice (Leprdb/db). de Luca et al. (13) further showed that selective restoration of neuronal leptin signaling in Leprdb/db mice using synapsin I (SynI)-Rb and neuron-specific enolase-Rb transgenes completely reversed the obese phenotype of Leprdb/db mice, reinforcing the notion that central leptin signaling plays a dominant role in leptin-mediated regulation of whole-body energy balance and metabolism.

Although strongly suggestive, studies in these mouse models do not rule out potential contributions to whole-body energy metabolism from leptin signaling in peripheral tissues. In the OBRSynIKO mouse, for example, it is uncertain whether the much milder obesity (compared with that of Leprdb/db mouse) is attributable to the incomplete deletion of neuronal Lepr or the presence of the normal peripheral leptin signaling in this model (12). Similarly, although neuron-specific transgenic expression of Syn-Rb and neuron-specific enolase-Rb appears to have "completely" reversed the obese phenotype of Leprdb/db mice, it is difficult to conclude that this is attributable to the lack of contributions from peripheral leptin signaling, because it is possible that the overexpression of Rb transgenes in multiple hypothalamic nuclei may result in overactivation of central leptin signaling, which may mask potential effects of lack of leptin signaling in the peripheral tissues of these mice (13).

Several studies, typically with examination of acute effects, have ascribed roles to direct leptin signaling in peripheral tissues. For example, direct leptin action in target tissues stimulates lipolysis and fatty acid oxidation in adipose tissues, skeletal muscle, and pancreas (15, 16, 17), decreases triglyceride content and secretion rates in liver (18), and suppresses insulin expression and secretion in pancreatic ß-cells (19, 20). How exactly the ascribed functions of direct leptin signaling in these peripheral tissues relate to long-term whole-body energy metabolism and glucose homeostasis in vivo remains unclear. Transgenic expression of an LEPR antisense RNA under the control of phosphoenolpyruvate carboxykinase promoter abrogates LEPR expression in white adipose tissue (WAT) and results in a phenotype characterized by obesity and whole-body insulin resistance (21). However, conditional knockout (KO) of leptin signaling in multiple peripheral tissues and physiological consequences of lacking peripheral leptin signaling as a whole have not been examined.

Work of several groups, including our own, have suggested that leptin feedback inhibits its own gene expression (22, 23, 24). Leptin administration decreases leptin mRNA expression in adipocytes (22); inactivation of the LEPR in Leprfa/fa rats is associated with increased leptin mRNA expression in adipose tissue and increased plasma leptin concentrations per unit fat mass (4, 23, 24). However, because deficiency of LEPR causes profound changes in food intake and body weight primarily through CNS-mediated mechanisms, which, in turn, have a strong effect on leptin gene expression (25), a direct role of adipocyte LEPR in the feedback suppression of leptin gene expression has not been established.

To directly assess the contribution of peripheral leptin signaling to the regulation of tissue and whole-body energy metabolism and to determine the importance of peripheral leptin signaling in the negative feedback regulation of leptin homeostasis, we used a tamoxifen (Tam)-inducible Cre-LoxP system to selectively disrupt leptin signaling only in peripheral tissues in a temporally controlled manner, i.e. conditional KO after weaning. This was achieved by selective deletion of exon 17, which encodes the signaling domain of LEPR, in peripheral tissues of the Leprflox/flox; ROSA26Cre-ERT2/+ mice by ip administration of a low dose of Tam. The regimen of Tam was sufficient to elevate local Tam levels in peripheral tissues, including liver, adipose tissue, small intestine, and pancreatic islets, to induce the nuclear translocation of cytoplasmic Cre-ERT2 fusion protein (26), resulting in the excision of the floxed exon 17 in these tissues. Because of the poor permeability of the blood-brain barrier to Tam and its metabolites (27), Cre-ERT2 fusion protein in the brain cells remains inactive (cytoplasmic) and thus the brain Lepr remains intact in Tam treated Leprflox/flox; ROSA26Cre-ERT2/+ mice (Cre-Tam mice). Using this mouse model, we show here that disruption of leptin signaling in peripheral tissues causes marked hyperleptinemia and posttranscriptional up-regulation of leptin production secondary to the inactivation of a "short-loop" negative feedback regulation in adipose tissue. However, the increased leptin production in adipose tissue is apparently counterbalanced by the increased circulating levels of a leptin binding protein, resulting in unaltered effective plasma leptin levels and normal levels of central leptin signaling activity. Furthermore, because Cre-Tam mice show no significant alterations in tissue and whole-body energy metabolism and insulin sensitivity, we conclude that peripheral leptin signaling as a whole plays at most a minor role in the regulation of energy metabolism in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of Leprflox/flox; ROSA26Cre-ERT2/+ mice
Cre-ERT2 fusion gene was introduced into the ROSA26 locus using a knock-in strategy (26, 28, 29). The targeting vector was constructed as follows: the coding sequence of Cre-ERT2 and the simian virus 40 (SV40) polyadenylation signal was excised from the pCre-ERT2 plasmid (a gift from Dr. Pierre Chambon, College de France, Paris, France) and inserted into an EcoRI-SalI digested pBSKII vector (Stratagene, La Jolla, CA) (26). The Cre-ERT2-SV40polyA fragment was excised with SpeI-SalI and inserted together with a XhoI-SacI fragment containing an Flp-recombinase recognition target (frt)-flanked puromycin expression cassette (frt-PGK promoter-puromycin-PGK polyA-frt), into a SpeI-SacI digested pBigT vector (30). The insert of the resulting plasmid, consisting of the adenoviral splice acceptor sequence followed by the Cre-ERT2, the SV40 polyadenylation signal, and the frt-flanked puromycin expression cassette, was excised by PacI-AscI and inserted into the ROSA26-PA vector (30). The final knock-in vector ROSA26Cre-ERT2-frt-puro-frt was linearized with KpnI and electroporated into 129/SV embryonic stem cells. Puromycin-resistant embryonic stem cell clones were analyzed by Southern blotting, and correctly targeted clones were injected into C57BL/6J blastocysts to generate germ-line transmitting chimeras. ROSA26Cre-ERT2/+ mice were first backcrossed for three generations to FVB/NJ mice and then backcrossed to Leprflox/flox (F/F) mice (98.5% FVB/NJ background) for two generations (31, 32). Leprflox/flox; ROSA26Cre-ERT2/+ mice were then mated with Leprflox/flox mice to generate Leprflox/flox; ROSA26Cre-ERT2/+ (Cre) and Leprflox/flox (nonCre) mice.

Genotyping and Tam administration
Cre and nonCre mice were identified by a PCR genotyping method using primers specific to Cre recombinase. Tam (1 mg/d per mouse) was injected ip for 5 d in 4- to 5-wk-old Cre and nonCre mice to generate Cre-Tam and control mice, respectively. Semiquantitative analyses of Tam-induced deletion of exon 17 in the tissues of Cre-Tam mice was performed using a multiplex PCR system (primers A, B, and C) as described previously (31). PCR products corresponding to Leprflox and Lepr{Delta}17 alleles were quantified using the Quantity One program (Bio-Rad, Hercules, CA).

Animal husbandry and phenotypic analyses
Mice were weaned at 3 wk of age, separated according to gender, and maintained in a barrier facility (12-h light, 12-h dark cycle) with ad libitum access to rodent breeder chow (PicoLab rodent chow 5058; PicoLab, St-Amable, Québec, Canada) and water. Daily food intake of individually housed mice was calculated from 72 or 96 h intake measures. Body composition was determined in anesthetized mice using dual-energy x-ray absorptiometry (Lunar PIXImus scanner; GE Medical Systems, Waukesha, WI). For glucose tolerance tests, blood glucose was measured at 0, 30, 60, and 90 min after a bolus ip glucose administration (1 mg/g body weight) to overnight-fasted mice. Blood glucose concentrations in the tail vein were measured using Glucometer Elite (Bayer, Elkhart, IN). Blood samples were collected either at basal (5-h food deprivation) or fasting condition (overnight fast), and plasma concentrations of leptin, insulin, adiponectin, and corticosterone were assayed by the Hormone Analysis Core, Mouse Metabolic Phenotyping Center (Vanderbilt University, Memphis, TN). Metabolic measurements were taken at the age of either 3 months [posttreatment day 60 (PTD 60)] or 9 months (PTD 240) at the specified (in parentheses) time points after Tam treatment, unless otherwise indicated. All animal experimentation described here was conducted in accord with accepted standards of humane animal care and has been approved by the Columbia University Institutional Animal Care and Use Committee.

Determination of leptin sensitivity
Overnight-fasted mice were injected with recombinant murine leptin (Amgen, Thousand Oaks, CA) (1 mg/kg, ip) or the same volume of saline. Levels of hypothalamic phosphorylated STAT3 (Try 705) (P-STAT3) and STAT3 before leptin injection and 30 min after the injection were determined by Western blotting. Phosphorylation of STAT3 and p44/42 MAPK was also determined in perigonadal adipose tissues (PG-AT). Antibodies to P-STAT3 (Try 705), phosphorylated p44/42 MAPK (Thr202/Tyr204) (P-MAPK), and MAPK (Cell Signaling Technology, Danvers, MA) and to STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for Western blotting as described previously (33). The fold increase in the ratios of P-STAT3/STAT3 and P-MAPK/MAPK before and after leptin treatment was used as an index for leptin sensitivity.

Quantitative RT-PCR analysis of gene expression
Quantitative PCR analysis was performed using Bio-Rad iQ SYBR Green Supermix in DNA Engine Opticon 2 real-time PCR detection system (Bio-Rad). Cyclophilin A was used as a control to normalize the initial RNA input. The {Delta}{Delta}Ct cycle threshold method was used to calculate relative expression levels (as ratio to cyclophilin A) in samples. PCR primers for measuring total Lepr transcripts (inclusive of all isoforms) were complementary to the region encoding the N-terminal sequence of LEPR; primers for total Rb transcripts [including both wild-type (wt) and mutant Rb] were complementary to exons 16 and 18b; primers for the wt Rb transcripts were complementary to exons 17 and 18b (7). All primer sequences used in this study are listed in supplemental Table 1Go (published as supplemental data on The Endocrine Society’s Journals Online web site at http://end.endojournals.org).


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  		TABLE 1. Body weight, nasoanal length, and basal plasma concentrations of hormones and metabolites in mice at age of 9 months, 8 months after Tam treatment (mean ± SD)

 
Leptin and adiponectin secretion assay
Secretion assays were performed as described previously (34). Briefly, PG-AT were washed and then incubated in Krebs-Ringer’s bicarbonate buffer supplemented with 1 g/liter glucose and 10 g/liter fatty acid-free BSA for 2 h. Leptin and adiponectin in the incubation media were assayed as in plasma samples. The rate of secretion was expressed as nanograms of leptin or micrograms of adiponectin secreted per gram of tissue per hour.

Determination of fractions of bound and free plasma leptin
The analysis was performed as described previously (35). Briefly, 100 µl plasma was mixed with 2 µl (0.18 ng/µl) tracer [125I]leptin (NEX340; PerkinElmer, Wellesley, MA) and incubated at 4 C overnight. Gel filtration was performed on a Superdex-200 column equilibrated and eluted with the same buffer [0.05 M NaPO4 and 0.15 M NaCl (pH 7.4)] at 4 C. Eighty 300-µl fractions were collected, and the amount of radioactivity in each fraction was counted in a COBRA II AUTO-GAMMA counter. The counts in the fractions were plotted to show the elution position of the bound leptin (first peak) and free leptin (second peak). The area under the curve of each peak was calculated using Prism 3 software (GraphPad Software, San Diego, CA) and was used to calculate the percentages of bound and free leptin in the plasma samples.

Statistical analysis
Statistical analyses were performed using Statistica version 6 (StatSoft, Tulsa, OK). All data are expressed as mean ± SD. ANOVA was used to assess the effects of attenuation of peripheral leptin signaling on various parameters in Cre-Tam mice vs. their same gender controls. Newman-Keuls test was used for post hoc comparisons when more than two groups were compared.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selective disruption of LEPR signaling in the peripheral tissues of Cre-Tam mice
Figure 1AGo depicts schematically the generation of the Cre-Tam mouse. Leprflox is a Lepr allele with exon 17 flanked by two loxP sequences (31, 32). To delete the floxed exon 17 in multiple peripheral tissues, a Cre-ERT2 fusion gene was introduced into ROSA26 locus to achieve a ubiquitous expression of Cre-ERT2, which is inactive until it is bound to its specific ligand, Tam (28, 29). ROSA26Cre-ERT2/+ mice were then backcrossed to Leprflox/flox mice to produce Leprflox/flox; ROSA26Cre-ERT2/+ (Cre mice) and Leprflox/flox (nonCre mice), segregating in the FVB/NJ background. Selective deletion of exon 17 in peripheral tissues was induced by ip administration of Tam (1 mg/d for 5 consecutive days) to 4- to 5-wk-old Cre mice (designated as Cre-Tam mice). Tam-treated nonCre littermates were used as controls.


Figure 1
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  		FIG. 1. Tam-induced selective deletion of exon 17 of Lepr in peripheral tissues of Leprflox/flox; ROSA26Cre-ERT2/+ mice. A, Schematic illustration of the generation of Cre-Tam mice via Tam-induced, Cre-ERT2-mediated deletion of exon 17 of Lepr. The relative positions and orientations of the loxP sites and primers A, B, and C, to exons 16, 17, and 17' of Lepr for the Leprflox and Lepr{Delta}17 alleles are shown. B, Representative results of PCR-based genotyping of Lepr in the tissues/organs of male (M) and female (F) Cre-Tam mice and a female Cre mouse (Cre). The mice were killed at PTD 90. Genomic DNA from brain (minus hypothalamus) (Bra), hypothalamus (Hyp), kidney (Kid), liver (Liv), pancreatic islets (PI), small intestine (Int), skeletal muscle (Mus), inguinal fat pad (Ing), perigonadal fat pad (PG), and intrascapular brown adipose tissue (BAT) were amplified for 30 cycles using primers A, B, and C. Tail DNA of an {Delta}17/F mouse was used as a control (Con) for amplification efficiencies of Leprflox and Lepr{Delta}17 alleles. C, The percentage of Leprflox allele remained in the tissue of Cre-Tam mice, expressed as intensity (Leprflox)/intensity (Leprflox + Lepr{Delta}17). The quantitative data were derived from mice killed between PTD 14 and PTD 240 (n = 12–17 for either gender).

 
The extent of Tam-induced deletion of exon 17 in various tissues of Cre-Tam mice was examined at multiple time points after Tam treatment (between PTD 14 and PTD 240) by gel electrophoresis and semiquantitative PCR (Fig. 1Go, B and C). Extensive deletion of exon 17 was detected in the liver (90%), PG-AT (85%), inguinal adipose tissue (73%), and small intestine (95%) of Cre-Tam mice. Less substantial deletions were also observed in pancreatic islets (60%), brown adipose tissue (BAT) (40%), skeletal muscle (20%), and kidney (25%). No deletion was detected in the hypothalamus or the rest of the brain in Cre-Tam mice, and no deletion occurred without Tam treatment (Fig. 1BGo). The deletion process was complete shortly after 5-d Tam treatment, and the pattern and extent of deletions were essentially unchanged between PTD 14 and PTD 240 (data not shown).

Deletion of exon 17 does not affect the expression or splicing of Lepr transcripts
To assess potential effects of deletion of exon 17 on the expression and splicing of Lepr transcripts, we examined the level and composition of Lepr transcripts by quantitative RT-PCR using respective primer sets that amplify the wt Rb isoform selectively, both the mutant and wt Rb isoform in combination, and all of the Lepr isoforms (Ra–Re) indiscriminately (Fig. 2Go) (primer sequences are shown in supplemental Table 1Go). The levels of the wt Rb transcript were reduced by approximately 88 and 93%, respectively, in PG-AT and liver of Cre-Tam mice but were not changed in the hypothalamus as expected (Fig. 2AGo). The levels of total Rb transcripts (the mutant and wt Rb combined) and total Lepr transcripts (all isoforms combined) in these tissues were not significantly different between Cre-Tam mice and controls (Fig. 2AGo). Consistent with the quantitative RT-PCR analyses, a near-complete conversion from wt Rb to the mutant Rb (Rb-{Delta}17) was evident in the PG-AT (Fig. 2BGo) and livers (data not shown) of Cre-Tam mice by gel analyses. Levels of the Re transcript, which is expressed at detectable levels only in PG-AT (6), were not significantly different between Cre-Tam mice and controls (Fig. 2CGo). Together, these data indicate that no significant alterations in Lepr splicing and in mRNA levels of Lepr isoforms occurred as a result of the deletion of exon 17 in the affected tissues.


Figure 2
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  		FIG. 2. Deletion of exon 17 has no effects on the expression level and splicing of Lepr transcripts. A, mRNA levels for the wt Rb isoform (wt), total Rb isoforms (include mutant and wt Rb), and all Lepr isoforms in PG-AT, liver, and hypothalamus (Hypo) of Cre-Tam mice and controls (Ctrl) killed at PTD 60 (n = 7–10). The values of control mice were defined as 1. **, P < 0.01, ***, P < 0.001, Cre-Tam vs. control mice. B, Gel analysis of RT-PCR products of PG-AT. C, mRNA levels for the Re isoform in PG-AT (n = 7).

 
Disruption of peripheral leptin signaling causes no significant changes in energy balance or glucose homeostasis
Phenotypic characterization of Cre-Tam mice was performed at various time points for up to 240 d after Tam treatment. No significant differences in energy balance and glucose metabolism, including body weight (Fig. 3AGo), adiposity (Fig. 3BGo), glucose tolerance (Fig. 3CGo), and growth (na-soanal length) (Table 1Go), were found between Cre-Tam mice and controls. These findings were further substantiated by indistinguishable morphologic appearances of PG-AT, liver, and BAT (Fig. 3DGo) and normal basal plasma concentrations of glucose, insulin, adiponectin, corticosterone, and nonesterified fatty acids (Table 1Go). Food intake (Fig. 4AGo) and hypothalamic neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA expression (Fig. 4BGo) were not altered in Cre-Tam mice. Finally, thermoregulation was also normal; Cre-Tam mice and the controls had similar core body temperatures (38.12 ± 0.14 vs. 38.09 ± 0.29 C) and responded similarly to a 2-h cold challenge at 4 C (37.20 ± 0.2 vs. 37.15 ± 0.23 C) (Fig. 4CGo). All of these results are consistent with unaltered, normal levels of CNS leptin signaling activity in Cre-Tam mice.


Figure 3
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  		FIG. 3. Cre-Tam mice have no abnormalities in energy metabolism and glucose homeostasis. A, Growth curves of male (M) and female (F) Cre-Tam mice and controls (Ctrl) (n = 7–17). B, Body composition determined at PTD 240 (n = 7–10). C, Glucose tolerance tests (1 mg/g wt) in female Cre-Tam mice and control mice at PTD 240 (n = 7–10). D, Representative hematoxylin-eosin-stained PG-AT, liver, and BAT tissue sections of female Cre-Tam and control mice at PTD 240.

 

Figure 4
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  		FIG. 4. Functions sensitive to central leptin signaling activity are unaltered in Cre-Tam mice. A, Food intakes of female Cre-Tam mice and the same-gender controls (Ctrl) at 3–10 wk after Tam treatment (n = 10–15). B and C, Hypothalamic mRNA levels of NPY and MCH and core body temperatures at room temperature (RT) and after a 2 h cold challenge at 4 C (Cold), respectively, in female Cre-Tam and control mice at PTD 240 (n = 7–10). M, Male; F, female.

 
Disruption of peripheral leptin signaling leads to hyperleptinemia
Despite the lack of changes in energy balance and glucose homeostasis, basal plasma leptin concentrations were markedly elevated in Cre-Tam mice: 5-fold (P < 0.01) and 8-fold (P < 0.001) increases in 9-month-old male and female mice, respectively, relative to the same-gender controls (Table 1Go). Similarly, in 3-month-old Cre-Tam mice and control mice, although fat mass was not significantly different (Fig. 5AGo), plasma leptin concentrations were 15.5 and 4.0 ng/ml, respectively, in the basal state, and 2.4 and 0.7 ng/ml in the fasted state (Fig. 5BGo), indicating that the phenotypic expression of hyperleptinemia in Cre-Tam mice was independent of age, weight, or gender. Plasma leptin concentrations were positively correlated with fat mass in both the Cre-Tam group and the control group, except that the slope is much steeper for the Cre-Tam group than for the control group (Fig. 5Go, C and D). Thus, despite the disproportionately high ratios of plasma leptin concentration to fat mass in Cre-Tam mice, plasma leptin levels remain responsive to changes in fat mass in these mice. Similarly, the responsiveness of plasma leptin levels to acute changes in energy fluxes (feeding and fasting) was also intact. These results suggest that disruption of leptin signaling in adipose tissue did not interfere with the functional regulation of plasma leptin levels in response to changes in adipose tissue mass (reflective of long-term energy balance) and in acute feeding status (short-term energy balance).


Figure 5
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  		FIG. 5. Disruption of peripheral leptin signaling leads to hyperleptinemia relative to fat mass. A and B, Body fat mass and basal and fasting plasma leptin concentrations, respectively, in female Cre-Tam mice and their controls (Ctrl) at PTD 60 (n = 5). C and D, Relationships between basal plasma leptin concentrations and fat mass in Cre-Tam and control mice at PTD 240 and PTD 60, respectively. Fat mass in A, C, and D was the combined weight of inguinal, perigonadal, retroperitoneal, and mesenteric fat pads. M, Male; F, female.

 
To confirm that hyperleptinemia in Cre-Tam mice was attributable to disruption of leptin signaling but not to the mere presence of the structurally mutated Lepr gene, we first measured leptin signaling in PG-AT, in which approximately 90% reduction in the wt Rb transcripts was achieved. Basal levels of both P-STAT3 and P-MAPK were present in the WT and Cre-Tam tissues. However, although the acute administration of exogenous leptin (1 mg/kg, ip) led to significant increases in P-STAT3 (1.5-fold above the basal level; P < 0.05) and P-MAPK (4.2-fold above the basal levels; P < 0.01) in the control adipose tissues, it caused no increase in the phosphorylation levels of either protein in the Cre-Tam adipose tissues (Fig. 6Go, A and B), consistent with a near-complete loss of leptin signaling activity in these tissues. We also compared ratios of plasma leptin to fat mass in littermates of the following two genotypes: F/F mice, which possess a normal leptin signaling system and have the structural integrity of Lepr, and Lepr{Delta}17/flox ({Delta}17/F) mice, which have near-normal leptin signaling but possess a copy of the structurally mutated Lepr. Although the heterozygous {Delta}17/F mice had slightly higher body fat mass and plasma leptin concentrations than the normal F/F mice, the plots for these parameters in both genotypes were on the same regression line (Fig. 6CGo). Plasma leptin concentrations per unit fat mass were not significantly different between F/F mice and {Delta}17/F mice (3.13 ± 1.11 vs. 3.51 ± 1.39 ng/ml·g), as opposed to the approximate 4-fold increase in Cre-Tam mice compared with their respective controls (Fig. 6DGo). Because {Delta}17/F mice are obligate heterozygotes for the mutant Lepr{Delta}17 allele and yet display the same plasma leptin to fat mass ratios as control F/F mice, this result also supports the conclusion that hyperleptinemia in Cre-Tam mice is not related to the presence of the mutant Lepr{Delta}17 allele beyond its effect on leptin signaling in peripheral tissues.


Figure 6
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  		FIG. 6. Disruption of peripheral leptin signaling but not the presence of the mutant LEPR ({Delta}17) causes hyperleptinemia. A, Representative Western blot results of basal and leptin-induced phosphorylation levels of STAT3 (Tyr705) and p44/42 MAPK (Thr202/Tyr204) in adipose tissues of Cre-Tam and control mice (PTD 60) measured before and 30 min after an ip leptin administration (1 mg/kg body weight). B, The average phosphorylation levels of P-STAT3 and P-MAPK in adipose tissue of two sets of male and three sets of female Cre-Tam and control mice. Data (mean ± SD) are expressed as ratios of P-STAT3/STAT3 and of P-MAPK/MAPK; all values are relative to that of control mice in the basal state, which is defined as 1. *, P < 0.05 and **, P < 0.01, respectively, basal vs. leptin stimulated. C, Relationship between basal plasma leptin concentrations and fat mass as determined by dual-energy x-ray absorptiometry in 10-wk-old female F/F and {Delta}17/F littermates (n = 6–7). D, Ratios of plasma leptin concentration to fat mass in 10-wk-old F/F and {Delta}17/F mice and in Cre-Tam and control mice (Ctrl) at PTD 60 (n = 5). The value of F/F or control mice was defined as 1. **, P < 0.01, Cre-Tam vs. controls.

 
Disruption of peripheral leptin signaling increases posttranscriptional leptin production in adipose tissue
To determine the cause of hyperleptinemia in the Cre-Tam mice, we measured leptin secretion rates and leptin mRNA levels in adipose tissue. At PTD 60, rates of leptin secretion in PG-AT were increased by 2.3-fold in Cre-Tam mice compared with controls (7.6 ± 1.4 vs. 3.3 ± 1.1 ng/g·h; P < 0.05) (Fig. 7AGo), whereas rates of adiponectin secretion were not altered (Fig. 7BGo). This increase in leptin secretion is independent of adiposity because fat mass was the same in Cre-Tam mice as in controls (Fig. 5AGo). Quantification of PG-AT leptin mRNA levels showed no differences between Cre-Tam and control mice, indicating that the increased leptin secretion rate in Cre-Tam mice was attributable to posttranscriptional regulation, a mechanism distinct from transcriptional regulation resulting from changes in metabolic fluxes (24, 25).


Figure 7
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  		FIG. 7. Peripheral leptin signaling regulates leptin protein expression posttranscriptionally. A and B, Rates of leptin and adiponectin secretion, respectively, in PG-AT of Cre-Tam mice and control mice at PTD 60 (n = 5). *, P < 0.05, Cre-Tam vs. controls. C, PG-AT mRNA levels for leptin (LEP), UCP3, ß3-AR, SREBP-1c, and CPT-1 in Cre-Tam and control mice at PTD 60 (n = 5–12).

 
To exclude the possibility that this posttranscriptional up-regulation of leptin expression may be attributed to unrecognized submorphological changes in adipocyte metabolism, we measured mRNA levels of several key metabolic genes, including uncoupling protein 3 (UCP3), ß3-adrenergic receptor (ß3-AR), sterol regulatory element binding protein-1c (SREBP-1c), and carnitine palmitoyl-transferease-1 (CPT-1) in PG-AT of Cre-Tam mice and controls. We found no differences in the expression levels of these genes (Fig. 7CGo), confirming that no major metabolic alterations occurred in the adipose tissue of Cre-Tam mice. Together, these results strongly suggest that hyperleptinemia in Cre-Tam mice via up-regulation of leptin secretion is caused by the disruption of direct leptin signaling in adipose tissue; these results revealed a short-loop negative feedback regulation that is distinct from the central leptin-mediated, metabolism-dependent "long-loop" negative feedback regulation, which inhibits primarily leptin mRNA expression.

Cre-Tam mice have increased plasma concentrations of total leptin but normal free leptin with normal central leptin signaling activities
The dissociation of hyperleptinemia from changes in tissue and whole-body energy metabolism in Cre-Tam mice was unexpected but raised the question whether the apparently normal energy metabolism in Cre-Tam mice was truly attributable to lack of effects associated with deletion of peripheral LEPR. The alternative explanation for the unaltered energy metabolism would be that this is a net result of two opposing effects from peripheral and central leptin actions, i.e. decreased energy expenditure and fatty acid oxidation attributable to disruption of peripheral LEPR and increased energy expenditure and fatty acid oxidation attributable to enhanced central leptin signaling caused by hyperleptinemia. To distinguish these two possibilities, we further assessed the status of central leptin signaling by measuring phosphorylation levels of hypothalamic STAT3 before and after leptin stimulation. Basal phosphorylation levels of STAT3 in the hypothalamus were identical between Cre-Tam mice and the controls (Fig. 8AGo) despite the marked difference in plasma leptin levels. Increases in STAT3 phosphorylation in response to acute administration of exogenous leptin were observed in both Cre-Tam mice and control mice (Fig. 8AGo). The same magnitude of increases suggests that Cre-Tam mice were equally sensitive to leptin as control mice. These results were in agreement with the unaltered food intake and hypothalamic NPY and MCH mRNA levels in Cre-Tam mice, indicating that the levels of central leptin signaling activity were unaffected and normal in Cre-Tam mice in the presence of apparent hyperleptinemia.


Figure 8
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  		FIG. 8. Cre-Tam mice display increased proportions of leptin bound to sLEPR and normal central leptin signaling activities. A, Basal and leptin-induced phosphorylation of hypothalamic STAT3 in Cre-Tam and control mice (PTD 60) measured before and 30 min after an ip leptin administration (1 mg/kg body weight) (n = 5). A representative Western blotting result (top) and the average ratios of P-STAT3 (Tyr705)/STAT3 in the hypothalami of two sets of male and three sets of female Cre-Tam and control mice (bottom). Data (mean ± SD) are expressed relative to the value of the control mice in the basal state, which is defined as 1. *, P < 0.05, basal vs. leptin stimulated. B, Proportions of bound and free leptin in plasma samples of four pairs of body fat content-matched Cre-Tam and control mice at PTD 60. Representative data from one pair of male and female mice were shown. The first peak is the elution position of the leptin-sLEPR complex, and the second peak represents free leptin (35 ). The percentage of bound to total plasma leptin is indicated.

 
To find the cause for the apparent dissociation between hyperleptinemia and central leptin signaling activities, we determined the bioavailability of plasma leptin in Cre-Tam mice. The percentage of bound and free leptin in plasma samples were quantified by a chromatographic method using radiolabeled leptin tracer. These experiments revealed that the percentage of bound leptin was markedly elevated in Cre-Tam mice relative to the controls: 87.2–92.9 vs. 33.8–49.8% (P < 0.05) in male mice and 95.7–96.2 vs. 70.2–79.2% (P < 0.05) in female mice (Fig. 8BGo). The elution position of the bound leptin by gel filtration is identical to that of soluble LEPR (sLEPR)-leptin complex, which is an inactive form of leptin as reported previously (35). Importantly, concentrations of free plasma leptin were indistinguishable between Cre-Tam mice and controls (0.45–1.23 vs. 0.36–1.78 ng/ml; P = 0.38). These results suggest that the increased plasma leptin in Cre-Tam mice is primarily sequestrated in an inactive pool, likely as leptin-sLEPR complexes, and provide an explanation for the lack of increase in central leptin signaling activities and metabolic changes in the presence of hyperleptinemia.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To assess the physiological role of peripheral leptin signaling and its relative contribution to energy metabolism in vivo, we created a conditional KO mouse model in which the signaling domain of LEPR was deleted at a near-completion level in several major peripheral tissues, including liver, adipose tissues, and small intestine in postweaning mice. We showed that disruption of peripheral leptin signaling resulted in marked hyperleptinemia, which is consistent with the inactivation of a feedback inhibition of leptin production in adipose tissue. We found no significant changes in body weight, adiposity, food intake, thermoregulation, and glucose homeostasis in Cre-Tam mice. The hyperleptinemia was associated with an increased fraction of bound leptin, which resulted in decreased relative leptin bioavailability and unchanged bioactive free leptin levels. We further showed that levels of leptin signaling via STAT3 in the hypothalamus were normal in Cre-Tam mice, and hypothalamic expression of NPY and MCH as well as food intake were unaffected. Thus, in the absence of alteration in central leptin signaling activity, lack of changes in tissue and whole-body energy balance in Cre-Tam mice strongly suggest that the metabolic effect of leptin signaling in the affected peripheral tissues is small or insignificant, in contrast to that in the CNS, despite the demonstrated acute metabolic effects attributable to direct leptin actions in these tissues in several reported short-term studies (15, 16, 17, 18, 19, 20). We must note that caution should be taken in interpreting the role of leptin in skeletal muscle, because only approximately 20% deletion of muscle Lepr was achieved in this model.

This conclusion is in full agreement with our previous findings that selectively restoring leptin signaling in the CNS of LEPR-null Leprdb/db mice is sufficient to reverse the obesity/diabetes phenotype of these mice (13). Ren et al. (36, 37) recently showed that Src homology 2 B1 (SH2B1) is an obligatory mediator of leptin signaling, and selective restoring of SH2B1 in the CNS of SH2B1–/– mice also resulted in normalization of leptin-mediated effects on energy balance and glucose metabolism. Our results were also consistent with the findings in the OBRAlbKO mice (liver-specific KO of LEPR) (12), in which loss of hepatic leptin signaling bears no consequence in hepatic or whole-body energy metabolism. However, our results appear to be different from the results obtained from two other mouse models, the TKO-OBR (for technical knockout of the OB receptor) mouse and Leprflox/flox RIPcre tg+ mouse (21, 38). The TKO-OBR mouse is deficient in LEPR expression in WAT attributable to the transgenic expression of an Lepr antisense RNA under the control of the phosphoenolpyruvate carboxykinase promoter (21). TKO-OBR mice are obese and insulin resistant. These mice have decreased energy expenditure with defective thermogenesis; they display decreased core body temperatures even in the absence of cold challenge. Huan et al. (21) postulate that attenuation of WAT leptin signaling in TKO-OBR mice lowers WAT ß3-AR expression levels, which in turn causes decreased thermogenesis and obese phenotype. The reason for the discrepancy between the antisense RNA model and our model is not entirely clear. However, we found that 90% deletion of the Rb isoform in adipose tissue was not associated with abnormal ß3-AR expression levels or impairment of thermoregulation in Cre-Tam mice. Although we cannot exclude the possibility that such abnormalities may ensue if a 100% deletion is achieved, it appears unlikely because the deletion at the current level proves to be very efficacious in increasing the rates of leptin secretion. Alternatively, it may be possible that there exists an alternative and yet unexplored mechanism that can explain the dramatic dysfunction in thermoregulation in the antisense RNA model. Leprdb/db mice, with global deficiency in LEPR activity, do display decreased core body temperatures. However, restoration of central leptin signaling alone corrected this defect (13), also suggesting that leptin signaling in adipose tissue is not essential for maintaining a normal core body temperature.

The Leprflox/flox RIPcre tg+ mouse bears a floxed exon17 of Lepr and expresses an active Cre recombinase under the control of the rat insulin promotor (RIP). The Leprflox/flox RIPcre tg+ mice exhibit similar levels of deletion of exon 17 in the pancreatic islets compared with Cre-Tam mice. However, unlike Cre-Tam mice, Leprflox/flox RIPcre tg+ mice develop abnormalities in insulin secretion, glucose intolerance, and obesity (38). Although deletion of exon 17 in Leprflox/flox RIPcre tg+ mice primarily occurs in pancreatic islets, leakages were reported, particularly in the brain tissue (38). Thus, it is uncertain whether the deletion of LEPR in the brain, albeit minor, may contribute to the phenotypic difference between Cre-Tam mice and Leprflox/flox RIPcre tg+ mice.

We and others have previously demonstrated the presence of negative feedback regulation of leptin gene expression. Leptin mRNA levels in adipose tissue are decreased after administration of exogenous leptin (22), whereas leptin mRNA levels and leptin secretion rates are increased when leptin signaling is disrupted as in Leprdb/db mice or Leprfa/fa rats (1, 23, 24, 39). Increasing central leptin signaling by intracerebroventricular leptin administration also decreases leptin mRNA expression in adipose tissue (40). This indirect effect of central leptin action on adipose tissue is likely mediated by changes in animal’s energy balance and reductions in energy fluxes in adipocytes (24). This negative feedback regulation via central action of leptin may be referred to as the long-loop negative feedback regulation. We have also shown that down-regulation of leptin mRNA expression in BAT by the long-loop feedback mechanism is predominantly mediated by an increased efferent sympathetic stimulation in this tissue (24).

In the present study, we have shown for the first time the existence of a short-loop negative feedback regulation of leptin production. Disruption of leptin signaling in adipose tissue results in marked increases in leptin secretion and hyperleptinemia in the absence of changes in central leptin signaling activity or tissue metabolic status. Furthermore, we show that this short-loop feedback regulation involves a posttranscriptional mechanism. Thus, it appears that leptin feedback inhibits its own gene expression and production through at least two distinct mechanisms: a central leptin signaling-mediated, tissue metabolism-dependent regulation of leptin mRNA expression (24) and an adipocyte LEPR-mediated, metabolism-independent posttranscriptional regulation of leptin protein synthesis and/or secretion.

An interesting finding in our study is that hyperleptinemia in Cre-Tam mice is associated with increased plasma levels of a leptin binding protein, which appears to be sLEPR based on the elution position from gel filtration chromatography. If the increased leptin binding protein is indeed sLEPR, this increase may be caused by a posttranscriptional mechanism because deletion of exon 17 is not associated with changes in Lepr mRNA expression levels and/or aberrant splicing in either liver or PG-AT of Cre-Tam mice. Increased ectodomain shedding of membrane-bound LEPR has been reported as a regulatory mechanism to control production of sLEPR (41, 42). Alternatively, increased plasma sLEPR levels are attributable to decreased clearance. In any case, increased plasma sLEPR levels result in increased leptin-sLEPR complexes and decreased leptin bioavailability (43). Our results support the general notion that leptin binding protein(s), such as sLEPR, functions to modulate bioavailability of plasma leptin, because Cre-Tam mice have a normal level of leptin signaling in the hypothalamus despite the markedly increased total and bound plasma leptin levels. The normal levels of central leptin signaling and energy metabolism in Cre-Tam mice agree well with the unaltered plasma levels of free leptin. Although molecular mechanisms for the up-regulation of plasma levels of the leptin binding protein requires additional study, our results indicate that disruption of leptin signaling in peripheral tissues alone is insufficient to cause changes in systemic energy balance. It is plausible that peripheral tissues are evolved to possess multiregulatory mechanisms, which in the absence of centrally initiated changes in leptin signaling, are orchestrated to preserve leptin homeostasis.

It remains to be determined whether increased plasma levels of the leptin binding protein in Cre-Tam mice are attributable to simultaneous disruption of leptin signaling in multiple peripheral tissues or simply increased leptin secretion in the absence of other metabolic changes. Chan et al. (44) reported that administration of pharmacological doses of leptin decreases plasma sLEPR levels in human, suggesting that leptin signaling may suppress sLEPR expression. Conversely, perfusion of rat liver with recombinant leptin induces a time- and dose-dependent formation of leptin-sLEPR complexes in the perfusate, secondary to increased hepatic production of sLEPR through a process that involves ectodomain shedding of the membrane-bound LEPR (41). Mouse models with exon 17 deletion of Lepr in adipocytes and hepatocytes alone may further help define specific roles of leptin signaling in the respective tissues regarding the production of leptin and leptin-binding protein(s) and their potential coordinated regulation by the two target tissues.

In summary, this study demonstrated that, compared with the central action of leptin, the role of leptin signaling in peripheral tissues as a whole is subordinate or insignificant in directing tissue energy metabolism in vivo. In the absence of potentially confounding changes in energy metabolism and central leptin signaling activity, Cre-Tam mice revealed a short-loop negative feedback mechanism in the regulation of leptin production that directly involves leptin signaling in adipocytes.


   Acknowledgments
 
We thank Dr. Rudy Leibel for many discussions during the study, Drs. Rudy Leibel and Domenico Accili for critical reviews of this manuscript, and Foster Chen for the assistance in genotyping.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK063034 (to Y.Z.), DK057621 (to S.C.), P30DK026687, and P30DK063068.

Present address for J.E.M.: Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, California 92121.

Present address for L.C.: Department of Medicine, Tongji Medical University, Wuhan, China.

Present address for C.L.: Department of Metabolic Disorders, Merck Research Laboratories, Rahway, New Jersey 07065.

Present address for S.C.: Department of Medicine, Albert Einstein College of Medicine, New York, New York 10461.

Disclosure Statement: J.E.M. received royalties from Takeda Pharmaceuticals. All other authors have nothing to declare.

First Published Online May 10, 2007

Abbreviations: ß3-AR, ß3-Adrenergic receptor; BAT, brown adipose tissue; CNS, central nervous system; CPT-1, carnitine palmitoyl-transferease-1; {Delta}17/F, Lepr{Delta}17/flox mice; F/F, Leprflox/flox mice; frt, Flp-recombinase recognition target; KO, knockout; LEPR, leptin receptor; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; P, phosphorylated; PG-AT, perigonadal adipose tissue; PTD, posttreatment day; RIP, rat insulin promotor; SH2B1, Src homology 2 B1; sLEPR, soluble LEPR; SREBP-1c, sterol regulatory element binding protein-1c; STAT, signal transducer and activator of transcription; SV40, simian virus 40; Syn, synapsin; Tam, tamoxifen; UCP3, uncoupling protein 3; WAT, white adipose tissue; wt, wild type.

Received February 26, 2007.

Accepted for publication May 1, 2007.


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 Introduction
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 Results
 Discussion
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