Tyrosine-dependent and -independent actions of leptin receptor in control of energy balance and glucose homeostasis

Lei Jiang; Jia You; Xinxin Yu; Lety Gonzalez; Yue Yu; Qiong Wang; Guoqing Yang; Wenjun Li; Cai Li; Yong Liu

doi:10.1073/pnas.0804589105

New Research In

Edited by Roger H. Unger, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 16, 2008
↵¹L.J. and J.Y. contributed equally to this work. (received for review May 13, 2008)

Abstract

Leptin regulates energy balance and glucose metabolism by activation of multiple signaling cascades mediated by the long-form leptin receptor Ob-Rb. However, the whole spectrum of signaling actions through the 3 cytoplasmic tyrosines of mouse Ob-Rb remains to be completely defined in vivo. Here, we generated 2 knockin lines of mice expressing mutant leptin receptors with phenylalanine substitution for all 3 tyrosines (Y123F) or for Tyr¹¹³⁸ alone (Y3F). Y123F animals developed overt obesity similar to that of Y3F animals with abrogated hypothalamic activation of STAT3 by leptin, but they exhibited more severe impairment in glucose tolerance. In striking contrast to db/db mice, however, both Y123F and Y3F mice showed attenuated adiposity with reduced hyperphagia, marked improvement in physical activity and adaptive thermogenesis, and significantly ameliorated glycemic control. Further, Y123F mice had hypothalamic neuropeptide Y/agouti-related protein expression maintained at prominently lower levels compared with db/db mice. Thus, these results provide direct physiological evidence that Ob-Rb exerts crucial metabolic actions not only through tyrosine-dependent, but also tyrosine-independent mechanisms in control of energy balance and glucose homeostasis.

Leptin is an adipocyte-derived hormone (1) that acts to regulate the balance between energy intake and expenditure primarily by hypothalamic activation of multiple signaling pathways through its long-form receptor Ob-Rb in leptin-responsive neurons (2, 3). Mice deficient in leptin (ob/ob) or functional leptin receptor (db/db) manifest obesity, hyperphagia, and hyperglycemia (4, 5). Although leptin resistance is found to be closely associated with obesity (6), the exact molecular defects underlying diminished leptin responsiveness remain poorly defined, largely ascribable to our incomplete understanding of the whole spectrum of Ob-Rb-mediated signaling events in energy homeostasis.

Leptin triggers activation of Janus kinase 2 (JAK2) by binding to Ob-Rb (7) and elicits an array of subsequent intracellular tyrosyl-phosphorylation-dependent signaling actions, thought to be mediated through the 3 cytoplasmic tyrosine residues (Tyr⁹⁸⁵, Tyr¹⁰⁷⁷, and Tyr¹¹³⁸) within mouse Ob-Rb. Phosphorylated Tyr¹¹³⁸ is known to recruit signal transducer and activator of transcription 3 (STAT3) and activate the crucial JAK2–STAT3 pathway in mediating leptin actions in energy homeostasis (8, 9). Phosphorylation at Tyr⁹⁸⁵ has been shown to mediate molecular events involving SH2-containing protein tyrosine phosphatase 2 (SHP2) (10, 11), extracellular signal-regulated kinase (ERK) (12, 13), and suppressor of cytokine signaling 3 (14, 15). However, only recently has an autoinhibitory role been suggested in vivo for Tyr⁹⁸⁵ in the l/l mice harboring a leucine substitution at this site (16). Whereas activation of other downstream signaling molecules, e.g., phosphatidylinositol 3-kinase (PI3K) (17) or STAT5 (18), may also contribute to the metabolic actions by Ob-Rb, the physiological importance of possible tyrosine-independent actions and their functional connections in vivo with tyrosine-dependent mechanisms remain largely unclear.

To understand fully the physiological contributions of phosphotyrosine-mediated signaling actions of Ob-Rb, we generated 2 lines of knockin mice by introducing tyrosine-to-phenylalanine substitution mutations simultaneously at all 3 intracellular tyrosine sites or at Tyr¹¹³⁸ alone. By comparing with db/db mice which are devoid of functional receptor signaling, we assessed the physiological importance of Ob-Rb signaling in the total absence of phosphotyrosine-mediated mechanisms, and actions mediated by Tyr⁹⁸⁵ and Tyr¹⁰⁷⁷ but in the absence of Tyr¹¹³⁸-mediated STAT3 cascade, in regulation of energy balance and glucose metabolism.

Results

Total Loss of Phosphotyrosine Actions of Ob-Rb Does Not Exacerbate the Obesity Phenotype Caused by Loss of STAT3 Action.

To maintain the structural integrity of Ob-Rb receptor, substitution mutations were introduced through homologous gene targeting, replacing with phenylalanine (F) the 3 tyrosine (Y) residues, Tyr⁹⁸⁵, Tyr¹⁰⁷⁷ and Tyr¹¹³⁸ (denoted Y123F), or Tyr¹¹³⁸ alone (denoted Y3F), within exon 18 of Ob-Rb (Fig. 1 A and B). Y123F and Y3F homozygotes were obtained after backcrossing for 6 generations onto the C57BL/6 background, and all subsequent studies were carried out compared with C57BL/6 db/db mice. The wild-type (WT) littermates used were derived from the knockin lines, which showed no differences in body weight (BW), or fed glucose levels compared with the age-matched WT littermates derived from C57BL/6 db/+ mice (data not shown), suggesting that little background difference existed metabolically between the two sources of experimental C57BL/6 animals. Quantitative RT-PCR analysis revealed comparable hypothalamic mRNA expression levels of Ob-Rb in Y123F and Y3F mice and in WT littermates, similar to that of the mutant Ob-Rb form in db/db animals [supporting information (SI) Fig. S1A]. Hypothalamic expression levels of Ob-Ra, one of the short isoforms of leptin receptor thought to lack signaling functions, were also similar in both knockin lines and WT littermates but dramatically lower than that in db/db mice because of the genetic mutation of the Ob-R gene (19). Similar to the reported findings in the s/s mice (8) with targeted substitution mutation at Tyr¹¹³⁸ by serine, replacement by phenylalanine of all 3 tyrosines or Tyr¹¹³⁸ within Ob-Rb abolished leptin-stimulated phosphorylation of STAT3 in the hypothalamus (Fig. 1C). Compared with db/db animals, however, both age- and sex-matched Y123F and Y3F mice showed lower obesity levels, greater snout–anus lengths, and interestingly, appreciably higher lean mass (Table 1 and Fig. S1B). In contrast to Y3F females that exhibited similarly impaired fertility (≈40% that of WT control) as found for the s/s females (8), Y123F female mice were completely infertile when bred with WT mates; however, neither Y3F nor Y123F male mice were able to reproduce over a 6-week period of breeding time (Table 1), indicating the importance of both STAT3-dependent and -independent functions of Ob-Rb in the control of reproduction.

Fig. 1.

Abrogation of hypothalamic STAT3 activation in Y3F and Y123F mice. (A) Schematic diagram showing the strategy of homologous gene targeting to replace exon 18 of the leptin receptor gene with the mutant exon 18 (Y123F) harboring phenylalanine (F) substitutions for all of the 3 tyrosine residues at positions 985, 1077, and 1138. The selection marker (LNL) cassette was subsequently removed by cre excision. The Y3F knockin mice were likewise generated. (B) Chimeric animals were obtained from targeted 129 embryonic stem cell clones, which were then used to generate WT (+/+), heterozygote (+/−), and homozygote (−/−) littermates. Shown is the successful gene targeting as confirmed by Southern blot analysis of PstI-digested genomic DNA from the tails of Y123F mice in the mixed 129Sv/C57BL/6 background, using an Ob-Rb-specific probe as indicated in A. The 5-kb fragment corresponds to the WT (+) allele, and the 3.5-kb fragment to the mutant allele (−). (C) Leptin-stimulated hypothalamic activation of STAT3 in WT littermates, Y3F, Y123F, and db/db mice. Age-matched male animals at 9–10 weeks of age (n = 4 or 5 for PBS or leptin treatment with each genotype) were fasted for 20 h and treated with vehicle or leptin at 2 mg/kg for 15 min by tail vein injection. The protein extracts of the dissected hypothalami were subjected to Western immunoblotting using the indicated antibodies.

View this table:

Table 1.

Phenotypic data for mice expressing mutant leptin receptor

Similar to C57BL/6 db/db mice, both homozygotic Y123F and Y3F animals developed early-onset obesity, exhibiting dramatic increases in BW gain starting from 4 weeks of age (Fig. 2A and Fig. S2A). However, the progressive BW increases in both lines started to decline considerably after 6 weeks of age compared with db/db animals, to an extent of ≈10% lower at 10 weeks (Fig. 2A) and between 16% and 30% lower at 28 weeks (data not shown). Of note, both male and female Y123F mice at 10 weeks of age exhibited slightly higher BW gain than their Y3F counterparts. Compared with WT male mice, body fat content of both lines was ≈7-fold higher at 8 weeks of age and ≈2-fold higher at 28 weeks of age. However, compared with db/db mice, the knockin lines weighed 30% less (Fig. 2B). For females at both ages, Y123F mice exhibited ≈4-fold higher fat accumulation than WT, but ≈15% lower than db/db animals (Fig. S2B); Y123F females at 8 weeks of age also displayed marginally but significantly higher fat mass than their Y3F counterparts. In addition, compared with WT littermates, male Y3F/+ and Y123F/+ heterozygotes at 8 weeks of age showed slight increases in BW gain similarly to that of db/+ animals and exhibited similar but much less pronounced degrees in the trend of adiposity as their homozygotic counterparts (Fig. S3). Moreover, both Y123F and Y3F homozygotes exhibited relatively modest hyperleptinemia compared with db/db animals (Table 1; 40% and 50% lower under fasted and fed conditions, respectively), although the exact impact of these Ob-Rb knockin mutations on circulating leptin levels remains to be determined in correlation with their adiposity levels. In parallel, Y123F and Y3F mice showed comparable hyperphagia but less pronounced (≈30% decreases in food intake) than db/db mice (Fig. 2C and Fig. S2C). These results thus reveal not only the importance of the Tyr¹¹³⁸-mediated STAT3 pathway, but also the crucial role of mechanism(s) independent of the 3 intracellular tyrosines, in control of adiposity and food intake.

Fig. 2.

Adiposity and energy intake. (A) BW changes of the Y3F and Y123F knockin mice, from 4 to 10 weeks of age, compared with WT and db/db mice on a chow diet. Shown are measurements for male mice (n > 10 per genotype). (B) Total body fat content was analyzed by NMR for male mice of each indicated genotype at 8 or 28 weeks of age (n > 10 per genotype). (C) Food intake was determined for male mice of each genotype at 7–8 weeks of age (n = 8∼10 per genotype). For all panels, data are shown as mean ± SEM. *, P < 0.05 vs. WT; #, P < 0.05 vs. db/db by ANOVA.

Dissociation of Adiposity and Glucose Homeostasis Between Y3F Mice and Y123F Mice.

Impairment in glucose homeostasis is highly correlated with obesity, and leptin has been proposed to regulate glucose metabolism through both adiposity-dependent and -independent pathways (20). Despite comparable levels of obesity at 10–13 weeks of age, homozygotic Y123F and Y3F mice showed considerable differences in the degree of hyperglycemia and insulin resistance states. Although both Y123F and Y3F animals had markedly lower fasted serum insulin and glucose levels than C57BL/6 db/db mice, Y123F animals developed a much higher degree of hyperinsulinemia than Y3F mice when fed ad libitum, similar to those observed in db/db mice (Table 1). Furthermore, glucose tolerance tests demonstrated that Y3F animals, similar to the reported s/s mice (21), had slightly impaired glucose tolerance, and Y123F mice displayed a degree of glucose intolerance markedly lower than db/db animals but significantly higher than their Y3F counterparts (Fig. 3A). Consistently, evaluation by homeostatic model assessment (HOMA) of insulin sensitivity (Fig. 3B) and insulin tolerance tests (Fig. S4) further revealed improved insulin sensitivity in Y3F mice than Y123F mice and than db/db mice. Notably, when normalized to or adjusted by covariate analysis for their respective body fat contents, HOMA assessment indicated appreciably improved insulin sensitivity in Y3F mice than Y123F animals (Fig. S5). Interestingly, Y123F and Y3F mice had similar circulating levels of resistin (another adipokine thought to play a causal role in insulin resistance) (22), largely correlating with their extents of adiposity (Table 1). Given the similar adiposity degrees of Y123F and Y3F mice, these results indicate the physiological importance of signaling through Tyr⁹⁸⁵ and Tyr¹¹⁰⁷ in the adiposity-independent regulation by leptin on glucose metabolism. However, because Y123F mice were leaner and displayed no significant improvement in adiposity-adjusted HOMA values compared with db/db animals (Fig. S5), the contribution of Ob-Rb tyrosine-independent mechanism(s) to systemic glycemic control may depend entirely on its regulation of adiposity.

Fig. 3.

Differing severity of glucose intolerance and insulin resistance in Y123F and Y3F mice. (A) Glucose tolerance tests were performed in male mice at 12 weeks of age for each indicated genotype (n = 7–8 per genotype), also shown as the calculated areas under curve (AUC). (B) HOMA of insulin sensitivity (IS) was determined for each genotype at 10–11 weeks of age (n = 5∼6 per genotype). HOMA = fasting glucose (millimolar) × fasting insulin (milliunits/liter)/22.5. For all panels, data are shown as mean ± SEM. *, P < 0.05 vs. WT; #, P < 0.05 vs. db/db; and †, P < 0.05 for Y123F vs. Y3F by ANOVA.

Effects of Ob-Rb Tyrosine Mutations on Energy Expenditure and Adaptive Thermogenesis.

We next investigated the effects of these knockin mutations in Ob-Rb on energy expenditure by using the comprehensive laboratory animal monitoring system (CLAMS). When normalized to their lean masses, both knockin lines and WT mice showed comparable oxygen consumption rates through a 12-h light/dark cycle, but all considerably lower than C57BL/6 db/db animals (Fig. S6). This is similar to documented findings (23) that argue against the validity of considering the lean mass solely as metabolically active when studying animals of dramatically different body sizes, e.g., WT vs. ob/ob mice. Therefore, we compared the metabolic rate by normalizing to the “metabolic size,” as reflected by BW^0.75, of each animal (24, 25). Both Y123F and Y3F mice displayed significantly lower oxygen consumption rates (by ≈20%) than WT animals (Fig. 4A). However, they exhibited similarly higher oxygen consumption rates than their db/db counterparts over the light cycle (Fig. 4A) or when assessed during periods of minimal activity (i.e., resting energy expenditure, REE) (Fig. 4B). Through both dark and light cycles, Y123F and Y3F mice showed markedly reduced physical activities (by ≈50%) compared with WT animals, but exhibited prominently higher activity levels (by 2- to 3-fold) than db/db mice (Fig. 4C). In parallel to these observations, the rectal temperatures of Y3F and Y123F animals were slightly but significantly higher than that of db/db mice (Table 1).

Fig. 4.

Effects of Ob-Rb intracellular tyrosine mutations on energy expenditure and adaptive thermogenesis. (A–C) Oxygen consumption (A), (REE) (B), and physical activity (C) were determined for 10- to 11-week-old male mice (n = 5–6 per genotype) and are shown as monitored through a 12-h light/dark cycle. (D) Adaptive thermogenesis was determined by a cold tolerance test in 12-week old male mice for each genotype. After food deprivation, rectal temperatures were measured at the indicated time points for mice placed in a cold vessel (n = 5–6 per genotype). (E) UCP1 and PGC1α mRNA levels in the BAT were analyzed for mice at the end of the test as indicated in D by real-time quantitative RT-PCR. For all panels, values are shown as mean ± SEM. *, P < 0.05 vs. WT; #, P < 0.05 vs. db/db by ANOVA.

To examine further the differences in energy expenditure among these animals, we performed a cold tolerance test to gauge adaptive thermogenesis (26), another major component of energy expenditure. During a 3-h exposure to cold, male Y123F and Y3F mice were similarly able to maintain their body temperature >32 °C, displaying slightly impaired thermogenesis compared with WT animals (Fig. 4D). By sharp contrast, db/db mice exhibited precipitous drop in body temperature, reaching <25 °C within 2.5-h exposure to cold. Moreover, cold-induced expression of uncoupling protein (UCP) 1 and peroxisome proliferator-activated receptor-γ coactivator (PGC) 1α in the brown adipose tissue (BAT), 2 key regulators in adaptive thermogenesis (27), was dramatically impaired in Y123F and Y3F mice compared with WT animals (Fig. 4E), whereas both exhibited significantly higher (>2-fold) expression levels than db/db mice. Female Y123F and Y3F animals also showed similar capacity for cold tolerance, with similar UCP1/PGC1α expression profiles detected in the BAT (Fig. S7). Therefore, tyrosine-independent actions by Ob-Rb play an essential role in regulation of adaptive thermogenesis, at least partially by control of UCP1/PGC1α expression in the BAT.

Impact of Ob-Rb Tyrosine Mutations on the Expression of Hypothalamic Neuropeptides.

To understand further the mechanistic actions that contribute to the differing phenotypes resulting from mutations of the Ob-Rb tyrosine residues, we analyzed the hypothalamic expression status of the orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), and anorexigenic peptides, proopiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) (28) in Y123F and Y3F animals under fed conditions. Compared with WT littermates, modest increases in NPY mRNA levels, but insignificant elevations in AgRP mRNA levels, were detected in both Y123F and Y3F mice. The expression level of AgRP in Y3F mice relative to WT mice was somewhat different from that reported for the s/s mice (8), and this discrepancy might either arise from the different knockin substitution mutation within Ob-Rb (i.e., Phe vs. Ser) or from the variability of AgRP expression measured in animals fed ad libitum. However, as in the s/s mice (8), prominent reductions in the expression of NPY and AgRP (by ≈50% and ≈70%, respectively) were observed in both knockin lines compared with db/db animals (Fig. 5 A and B). Similar to db/db mice, the expression of POMC and CART in Y123F and Y3F mice was significantly reduced compared with WT animals, with 40–60% decreases seen for POMC and ≈30% reductions for CART (Fig. 5 C and D). Both leptin and insulin are known to regulate the expression of these neuropeptides (29). Because Y123F mice presumably had lower ambient leptin and comparable ambient insulin levels compared with db/db mice (corresponding to their levels of hyperleptinemia and hyperinsulinemia), these data indicate that the Ob-Rb receptor with mutations of all 3 tyrosine residues probably retains most of its capacity in suppression of NPY and AgRP expression, implying a likely predominant role for Ob-Rb tyrosine-independent mechanism(s) in regulation of the NPY/AgRP neurons. However, given the overt hyperleptinemia that failed to stimulate POMC/CART expression in Y3F mice compared with WT littermates (Table 1), Tyr¹¹³⁸-mediated STAT3 pathway may mainly act to regulate the POMC/CART neurons, in accordance with previously reported studies (8).

Fig. 5.

Hypothalamic expression of neuropeptides in WT, Y3F, Y123F, and db/db mice. The mRNA levels of NPY (A), AgRP (B), POMC (C), and CART (D) were determined by quantitative RT-PCR for 12-week-old male mice fed ad libitum (n = 6–9 for each genotype), with cyclophilin used as an endogenous control. Data are shown as mean ± SEM. *, P < 0.05 vs. WT; #, P < 0.05 vs. db/db by ANOVA.

Discussion

The aim of the present work was to determine the roles of intracellular tyrosine phosphorylation in mediating the physiological functions of Ob-Rb in vivo. Because all 3 tyrosines within the intracellular domain of Ob-Rb are phosphorylated upon leptin activation, the triple-knockin mice devoid of tyrosine phosphorylation can provide critical insights on the role of tyrosine phosphorylation-independent actions of Ob-Rb in mediating the biological effects of leptin. Surprisingly, our results demonstrate that in the absence of tyrosine phosphorylation, the metabolic actions of leptin are not abolished, highlighting the need to determine the contributions of these actions.

Our findings are summarized as follows. First, similar to the reported s/s mice (8, 9, 21), abrogation of Tyr¹¹³⁸-dependent STAT3 signaling in Y3F mice led to severe obesity associated with deregulated energy balance and marginally impaired glucose tolerance. Moreover, compared with the findings in the s/s mice, Y3F animals displayed similar impairments in female fertility, physical activities, and adaptive thermogenesis, and similar hypothalamic NPY and POMC expression patterns. Second, although exhibiting little impact on the regulation of BW and energy balance, actions through Tyr⁹⁸⁵ and Tyr¹⁰⁷⁷ contributed significantly to systemic glycemic control. This finding illustrates a direct regulatory action by leptin in glucose homeostasis, not arising from the secondary effects of leptin signaling on adiposity. These results are in line with the pair-feeding studies in the s/s mice, which exhibit lower blood glucose than db/db mice despite similar BW (21). In addition, it is worth noting that Tyr⁹⁸⁵- and/or Tyr¹⁰⁷⁷-mediated signaling mechanisms are involved in the control of reproduction, as exhibited by the total loss of fertility in Y123F females. Third, but most importantly, the substantial amelioration in Y123F mice both in adiposity and glucose intolerance compared with db/db mice clearly demonstrates a critical role for additional tyrosine-independent mechanism(s) by Ob-Rb in controlling a broad spectrum of metabolic aspects, ranging from food intake, physical activity, adaptive thermogenesis, to glucose metabolism. Taken together, our findings not only demonstrate the essential role of tyrosine-dependent pathways mediated by Ob-Rb, but also provide direct physiological evidence that tyrosine-independent mechanism(s) exerts crucial metabolic functions in control of energy balance and glucose homeostasis.

This work provides an in vivo model showing the physiological contributions of signaling through Tyr⁹⁸⁵ and Tyr¹⁰⁷⁷, e.g., activation of either the SHP2–ERK or STAT5 pathway (18), or both, to regulation of glucose metabolism. Moreover, this work underscores the metabolic importance of tyrosine-independent actions by Ob-Rb in exerting the broad physiological functions of leptin, of which the exact molecular signaling events remain to be delineated. Mechanistically, this can be, at least in part, attributed to down-regulation of the hypothalamic NPY/AgRP neuropeptides in a manner independent of Ob-Rb tyrosine-mediated mechanisms. Because leptin has been shown to regulate NPY and AgRP by PI3K activation (30), it is tempting to speculate that a central JAK2–PI3K cascade is likely one of the key components (17, 31). In addition, other modulators of Ob-Rb signaling pathways, e.g., SH2-B (32), may also serve as important players. Given the central importance of leptin function in metabolic control through actions in the hypothalamus and in the periphery (33–35), further molecular dissection of the detailed interplay between these multiple signaling components, especially under pathophysiologic conditions, will lead us to understand better the molecular impairments responsible for the occurrence of leptin resistance that underlie the pathogenesis of human obesity and type 2 diabetes.

Materials and Methods

Mice.

Generation of knockin mice.

The gene targeting and generation of chimeric mice were carried out at the Transgenic Technology Center of University of Texas Southwestern Medical Center, Dallas. To generate knockin mice expressing Ob-Rb^{F985/1077/1138} (Y123F) and Ob-Rb^F1138 (Y3F), targeting vectors were constructed by introducing the lox-flanked neomycin resistance gene, Lox-Neo-Lox (LNL), downstream of the mutant exon 18 of the Ob-Rb gene that harbor the targeted substitution mutations for Y123F and Y3F, respectively (Fig. 1A). The targeting DNA constructs were transfected into 129/Sv embryonic stem (ES) cells by electroporation, and stable homologous recombinant clones were selected with G418. Exon 18 was amplified by PCR from targeted ES cell clones and sequenced to confirm Tyr-to-Phe substitutions. Confirmed ES clones were then injected into blastocysts and transferred into pseudopregnant females to generate chimeric agouti mice. Successful gene targeting was subsequently confirmed by Southern blotting. To remove the LNL selection cassette, germ-line-transmitted knockin mice were crossed with protamine-Cre mice (36), removing LNL from the male germ line only.

Backcross breeding and animal care.

LNL-deleted heterozygote Y123F and Y3F animals in the mixed 129Sv/C57BL/6 background were backcrossed to the WT C57BL/6 mice (from Shanghai Laboratory Animals Co.) for 6 generations to obtain mice >99% in the C57BL/6 background. Heterozygote mice were subsequently intercrossed to yield homozygotes and WT littermates, and the mutation sites were confirmed again by direct sequencing of Ob-Rb RT-PCR products derived from hypothalamic RNA (data not shown). C57BL/6 db/+ mice (from Model Animal Research Center of Nanjing University) were used for breeding to generate db/db animals. Mice were housed at a temperature of 22 ± 3 °C under a 12-h dark/light cycle (lights on at 6:30 AM) in accredited animal facilities of Shanghai Institute for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), and University of Texas Southwestern Medical Center (UTSW), with ad libitum access to standard chow and water. Animals for study were weaned at 22 days of age, and all experimental procedures and protocols were approved by the Institutional Animal Care and Use Committees at the Institute for Nutritional Sciences, SIBS, CAS, and UTSW.

Physiologic Phenotyping.

The BW of animals was monitored weekly for each genotype and sex of mice from 4 to 28 weeks of age. Body fat and lean mass were measured by NMR with a Minispec Mq7.5 Analyzer (Bruker). Food consumption was measured for mice at 7–8 weeks of age individually caged by weighing food daily before the dark cycle for 1 week. For fertility studies of each genotype, females at 9 or 18 weeks of age and males at 9 weeks of age were individually caged with age-matched WT mates, respectively. Pup delivery was monitored as the fertility score for a period of 6 weeks. Fasting glucose was determined in blood from tail vein of male mice at 9 and 10 weeks of age after a 6-h (8:30–14:30) fasting, by using a glucometer (FreeStyle). For blood measurements, mice at 10–11 weeks of age were anesthetized and killed, either overnight fasted (16 h) or fed ad libitum. Before killing, snout–anus lengths were measured with a vernier caliper. Blood was collected from heart. Levels of insulin, leptin, and resistin were analyzed with a mouse serum adipokine LINCOplex kit (Linco Research) with the Bio-Plex System (Bio-Rad) according to the manufacturer's instructions.

Glucose and Insulin Tolerance Tests and HOMA.

A glucose tolerance test was performed in male mice at 12 weeks of age after a 6-h (8:30–14:30) fast. Glucose concentrations were measured in blood collected by venous bleeding from tail vein, immediately before and 30, 60, and 120 min after a bolus i.p. injection of glucose at 0.75 g/kg. An insulin tolerance test was performed in 4-h (9:00–13:00) fasted male mice at 13 weeks of age. Glucose concentrations were likewise measured by venous bleeding at 0, 15, 30, 60, and 120 min after i.p. injection with human insulin (Eli Lilly) at 2 units/kg. HOMA values were derived from fasting glucose (millimolar) × fasting insulin (milliunits/liter)/22.5.

Metabolic Rate and Physical Activity.

Oxygen consumption and physical activity were determined for male mice fed ad libitum at 10–11 weeks of age by using CLAMS (Columbus Instruments) according to the manufacturer's instructions. Animals were acclimated to the system for 16–20 h, and measurement of VO₂ was done during the next 24 h. Voluntary activity was derived from the x axis beam breaks monitored every 15 min. REE was analyzed by calculating the oxygen consumption over the resting period, during which the x axis beam breaks were <20 per h.

Cold Tolerance Test.

A cold tolerance test was performed in 12-week-old mice. Body temperature was measured at room temperature at 9:00 AM for mice fed ad libitum by using microprobe thermometer (Physitemp Instruments). Animals were then subjected to cold at the bottom of 1-L glass beakers submerged in ice water without access to food or water. Rectal temperature was measured at the indicated time after exposure to cold.

Real-Time Quantitative RT-PCR Analysis.

Mice examined in the cold tolerance test for each genotype were killed at the end of the cold exposure or when their body temperature fell <20 °C, and the BAT was removed and snap-frozen immediately in liquid nitrogen for subsequent RNA extraction. Real-time quantitative PCR was performed with ABI Prism 7500 sequence detection system by following the manufacturer's recommendations (Applied Biosystems), using the desired primers (Table S1).

Leptin-Induced Hypothalamic STAT3 Activation.

Mice at 9–10 weeks of age were fasted for 20 h to suppress endogenous leptin levels. After deep anesthetization with sodium pentobarbital, mice were treated with vehicle (PBS) or recombinant mouse leptin (National Hormone and Peptide Program, UCLA) at 2 mg/kg for 15 min by tail vein injection. Hypothalami were dissected on ice-cold plates and quickly frozen in liquid nitrogen, and protein extracts were prepared by homogenizing with RIPA lysis buffer. Hypothalamic protein extracts (≈40 μg) were subjected to separation by SDS/PAGE followed by transfer to PVDF filter membranes (Amersham Biosciences). The filters were subsequently blotted with antibody against phospho-STAT3 (pTyr705), and the same blots were analyzed with anti-STAT3 antibody (Cell Signaling) after stripping.

Statistical Analysis.

All data were presented as mean values ± SEM. Comparisons between all groups were assessed by 1-way ANOVA with P < 0.05 considered statistically significant.

Acknowledgments

We thank J. M. Friedman (Rockefeller University) for initiation of the project, J. Repa (University of Texas Southwestern Medical School, Dallas) for animal care and handling, J. Shen [Institute for Nutritional Sciences, Chinese Academy of Sciences (CAS), Shanghai] for assistance with CLAMS, X. Wang (Duke University, Durham, NC) for insightful discussions, and Z. Liu (National Cancer Institute, National Institutes of Health) for critical reading of the manuscript. This work was supported by The Ministry of Science and Technology 973 Program 2006CB503900, National Natural Science Foundation Grants 90713027 and 30728024, CAS Knowledge Innovation Programs KSCX1-YW-02 and KSCX2-YW-R-115, One Hundred Talents Program, and the CAS/State Administration of Foreign Experts International Partnership Program and Science and Technology Commission of Shanghai Municipality Grants 04DZ14007 and 07JC14011 (to Y.L.) and National Institutes of Health Grant R01-DK60137 (to C.L.).

Footnotes

³To whom correspondence may be sent at the present address:
Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065.
E-mail: cai_li{at}merck.com
⁴To whom correspondence may be addressed. E-mail: liuy{at}sibs.ac.cn
Author contributions: W.L., C.L., and Y.L. designed research; L.J., J.Y., X.Y., L.G., Y.Y., Q.W., and G.Y. performed research; L.J., J.Y., C.L., and Y.L. analyzed data; and L.J., J.Y., C.L., and Y.L. wrote the paper.
↵²Present address: Biomedical Sciences Institute, Agency for Science, Technology and Research, Singapore.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0804589105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA

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(2004) LRb-STAT3 signaling is required for the neuroendocrine regulation of energy expenditure by leptin. Diabetes 53:3067–3073.
OpenUrl Abstract/FREE Full Text
↵
1. Li C,
2. Friedman JM
(1999) Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction. Proc Natl Acad Sci USA 96:9677–9682.
OpenUrl Abstract/FREE Full Text
↵
1. Zhang EE,
2. Chapeau E,
3. Hagihara K,
4. Feng GS
(2004) Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism. Proc Natl Acad Sci USA 101:16064–16069.
OpenUrl Abstract/FREE Full Text
↵
1. Bjorbaek C,
2. et al.
(2001) Divergent roles of SHP-2 in ERK activation by leptin receptors. J Biol Chem 276:4747–4755.
OpenUrl Abstract/FREE Full Text
↵
1. Banks AS,
2. Davis SM,
3. Bates SH,
4. Myers MG, Jr
(2000) Activation of downstream signals by the long form of the leptin receptor. J Biol Chem 275:14563–14572.
OpenUrl Abstract/FREE Full Text
↵
1. Bjorbaek C,
2. El-Haschimi K,
3. Frantz JD,
4. Flier JS
(1999) The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem 274:30059–30065.
OpenUrl Abstract/FREE Full Text
↵
1. Bjorbaek C,
2. et al.
(2000) SOCS3 mediates feedback inhibition of the leptin receptor via Tyr-985. J Biol Chem 275:40649–40657.
OpenUrl Abstract/FREE Full Text
↵
1. Bjornholm M,
2. et al.
(2007) Mice lacking inhibitory leptin receptor signals are lean with normal endocrine function. J Clin Invest 117:1354–1360.
OpenUrl CrossRef PubMed
↵
1. Niswender KD,
2. et al.
(2001) Intracellular signalling: Key enzyme in leptin-induced anorexia. Nature 413:794–795.
OpenUrl
↵
1. Gong Y,
2. et al.
(2007) The long form of the leptin receptor regulates STAT5 and ribosomal protein S6 via alternate mechanisms. J Biol Chem 282:31019–31027.
OpenUrl Abstract/FREE Full Text
↵
1. Lee GH,
2. et al.
(1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632–635.
OpenUrl CrossRef PubMed
↵
1. Kamohara S,
2. Burcelin R,
3. Halaas JL,
4. Friedman JM,
5. Charron MJ
(1997) Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389:374–377.
OpenUrl CrossRef PubMed
↵
1. Bates SH,
2. Kulkarni RN,
3. Seifert M,
4. Myers MG, Jr
(2005) Roles for leptin receptor/STAT3-dependent and -independent signals in the regulation of glucose homeostasis. Cell Metab 1:169–178.
OpenUrl CrossRef PubMed
↵
1. Steppan CM,
2. et al.
(2001) The hormone resistin links obesity to diabetes. Nature 409:307–312.
OpenUrl CrossRef PubMed
↵
1. Breslow MJ,
2. et al.
(1999) Effect of leptin deficiency on metabolic rate in ob/ob mice. Am J Physiol 276:E443–E449.
OpenUrl PubMed
↵
1. Himms-Hagen J
(1997) On raising energy expenditure in ob/ob mice. Science 276:1132–1133.
OpenUrl FREE Full Text
↵
1. Hwa JJ,
2. et al.
(1997) Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice. Am J Physiol 272:R1204–R1209.
OpenUrl PubMed
↵
1. Lowell BB,
2. Spiegelman BM
(2000) Towards a molecular understanding of adaptive thermogenesis. Nature 404:652–660.
OpenUrl PubMed
↵
1. Puigserver P,
2. et al.
(1998) A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92:829–839.
OpenUrl CrossRef PubMed
↵
1. Morton GJ,
2. Cummings DE,
3. Baskin DG,
4. Barsh GS,
5. Schwartz MW
(2006) Central nervous system control of food intake and body weight. Nature 443:289–295.
OpenUrl CrossRef PubMed
↵
1. Arora S,
2. Anubhuti
(2006) Role of neuropeptides in appetite regulation and obesity: A review. Neuropeptides 40:375–401.
OpenUrl CrossRef PubMed
↵
1. Morrison CD,
2. Morton GJ,
3. Niswender KD,
4. Gelling RW,
5. Schwartz MW
(2005) Leptin inhibits hypothalamic NPY and AgRP gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling. Am J Physiol 289:E1051–E1057.
OpenUrl
↵
1. Morton GJ,
2. et al.
(2005) Leptin regulates insulin sensitivity via phosphatidylinositol-3-OH kinase signaling in mediobasal hypothalamic neurons. Cell Metab 2:411–420.
OpenUrl CrossRef PubMed
↵
1. Ren D,
2. Li M,
3. Duan C,
4. Rui L
(2005) Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice. Cell Metab 2:95–104.
OpenUrl CrossRef PubMed
↵
1. Minokoshi Y,
2. et al.
(2002) Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339–343.
OpenUrl CrossRef PubMed
↵
1. Unger RH
(2004) The hyperleptinemia of obesity: Regulator of caloric surpluses. Cell 117:145–146.
OpenUrl CrossRef PubMed
↵
1. Wang MY,
2. et al.
(2008) Adipogenic capacity and the susceptibility to type 2 diabetes and metabolic syndrome. Proc Natl Acad Sci USA 105:6139–6144.
OpenUrl Abstract/FREE Full Text
↵
1. O'Gorman S,
2. Dagenais NA,
3. Qian M,
4. Marchuk Y
(1997) Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells. Proc Natl Acad Sci USA 94:14602–14607.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 105 (47)

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[1] ↵
Zhang Y,
et al.
(1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432.
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[2] Zhang Y,

[3] et al.

[4] ↵
Campfield LA,
Smith FJ,
Guisez Y,
Devos R,
Burn P
(1995) Recombinant mouse OB protein: Evidence for a peripheral signal linking adiposity and central neural networks. Science 269:546–549.
OpenUrl Abstract/FREE Full Text

[5] Campfield LA,

[6] Smith FJ,

[7] Guisez Y,

[8] Devos R,

[9] Burn P

[10] ↵
Cohen P,
et al.
(2001) Selective deletion of leptin receptor in neurons leads to obesity. J Clin Invest 108:1113–1121.
OpenUrl CrossRef PubMed

[11] Cohen P,

[12] et al.

[13] ↵
Friedman JM,
Halaas JL
(1998) Leptin and the regulation of body weight in mammals. Nature 395:763–770.
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[14] Friedman JM,

[15] Halaas JL

[16] ↵
Chua SC, Jr,
et al.
(1996) Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 271:994–996.
OpenUrl Abstract/FREE Full Text

[17] Chua SC, Jr,

[18] et al.

[19] ↵
Mantzoros CS,
Flier JS
(2000) Leptin as a therapeutic agent: Trials and tribulations. J Clin Endocrinol Metab 85:4000–4002.
OpenUrl CrossRef PubMed

[20] Mantzoros CS,

[21] Flier JS

[22] ↵
Kloek C,
et al.
(2002) Regulation of Jak kinases by intracellular leptin receptor sequences. J Biol Chem 277:41547–41555.
OpenUrl Abstract/FREE Full Text

[23] Kloek C,

[24] et al.

[25] ↵
Bates SH,
et al.
(2003) STAT3 signalling is required for leptin regulation of energy balance but not reproduction. Nature 421:856–859.
OpenUrl CrossRef PubMed

[26] Bates SH,

[27] et al.

[28] ↵
Bates SH,
et al.
(2004) LRb-STAT3 signaling is required for the neuroendocrine regulation of energy expenditure by leptin. Diabetes 53:3067–3073.
OpenUrl Abstract/FREE Full Text

[29] Bates SH,

[30] et al.

[31] ↵
Li C,
Friedman JM
(1999) Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction. Proc Natl Acad Sci USA 96:9677–9682.
OpenUrl Abstract/FREE Full Text

[32] Li C,

[33] Friedman JM

[34] ↵
Zhang EE,
Chapeau E,
Hagihara K,
Feng GS
(2004) Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism. Proc Natl Acad Sci USA 101:16064–16069.
OpenUrl Abstract/FREE Full Text

[35] Zhang EE,

[36] Chapeau E,

[37] Hagihara K,

[38] Feng GS

[39] ↵
Bjorbaek C,
et al.
(2001) Divergent roles of SHP-2 in ERK activation by leptin receptors. J Biol Chem 276:4747–4755.
OpenUrl Abstract/FREE Full Text

[40] Bjorbaek C,

[41] et al.

[42] ↵
Banks AS,
Davis SM,
Bates SH,
Myers MG, Jr
(2000) Activation of downstream signals by the long form of the leptin receptor. J Biol Chem 275:14563–14572.
OpenUrl Abstract/FREE Full Text

[43] Banks AS,

[44] Davis SM,

[45] Bates SH,

[46] Myers MG, Jr

[47] ↵
Bjorbaek C,
El-Haschimi K,
Frantz JD,
Flier JS
(1999) The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem 274:30059–30065.
OpenUrl Abstract/FREE Full Text

[48] Bjorbaek C,

[49] El-Haschimi K,

[50] Frantz JD,

[51] Flier JS

[52] ↵
Bjorbaek C,
et al.
(2000) SOCS3 mediates feedback inhibition of the leptin receptor via Tyr-985. J Biol Chem 275:40649–40657.
OpenUrl Abstract/FREE Full Text

[53] Bjorbaek C,

[54] et al.

[55] ↵
Bjornholm M,
et al.
(2007) Mice lacking inhibitory leptin receptor signals are lean with normal endocrine function. J Clin Invest 117:1354–1360.
OpenUrl CrossRef PubMed

[56] Bjornholm M,

[57] et al.

[58] ↵
Niswender KD,
et al.
(2001) Intracellular signalling: Key enzyme in leptin-induced anorexia. Nature 413:794–795.
OpenUrl

[59] Niswender KD,

[60] et al.

[61] ↵
Gong Y,
et al.
(2007) The long form of the leptin receptor regulates STAT5 and ribosomal protein S6 via alternate mechanisms. J Biol Chem 282:31019–31027.
OpenUrl Abstract/FREE Full Text

[62] Gong Y,

[63] et al.

[64] ↵
Lee GH,
et al.
(1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632–635.
OpenUrl CrossRef PubMed

[65] Lee GH,

[66] et al.

[67] ↵
Kamohara S,
Burcelin R,
Halaas JL,
Friedman JM,
Charron MJ
(1997) Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389:374–377.
OpenUrl CrossRef PubMed

[68] Kamohara S,

[69] Burcelin R,

[70] Halaas JL,

[71] Friedman JM,

[72] Charron MJ

[73] ↵
Bates SH,
Kulkarni RN,
Seifert M,
Myers MG, Jr
(2005) Roles for leptin receptor/STAT3-dependent and -independent signals in the regulation of glucose homeostasis. Cell Metab 1:169–178.
OpenUrl CrossRef PubMed

[74] Bates SH,

[75] Kulkarni RN,

[76] Seifert M,

[77] Myers MG, Jr

[78] ↵
Steppan CM,
et al.
(2001) The hormone resistin links obesity to diabetes. Nature 409:307–312.
OpenUrl CrossRef PubMed

[79] Steppan CM,

[80] et al.

[81] ↵
Breslow MJ,
et al.
(1999) Effect of leptin deficiency on metabolic rate in ob/ob mice. Am J Physiol 276:E443–E449.
OpenUrl PubMed

[82] Breslow MJ,

[83] et al.

[84] ↵
Himms-Hagen J
(1997) On raising energy expenditure in ob/ob mice. Science 276:1132–1133.
OpenUrl FREE Full Text

[85] Himms-Hagen J

[86] ↵
Hwa JJ,
et al.
(1997) Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice. Am J Physiol 272:R1204–R1209.
OpenUrl PubMed

[87] Hwa JJ,

[88] et al.

[89] ↵
Lowell BB,
Spiegelman BM
(2000) Towards a molecular understanding of adaptive thermogenesis. Nature 404:652–660.
OpenUrl PubMed

[90] Lowell BB,

[91] Spiegelman BM

[92] ↵
Puigserver P,
et al.
(1998) A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92:829–839.
OpenUrl CrossRef PubMed

[93] Puigserver P,

[94] et al.

[95] ↵
Morton GJ,
Cummings DE,
Baskin DG,
Barsh GS,
Schwartz MW
(2006) Central nervous system control of food intake and body weight. Nature 443:289–295.
OpenUrl CrossRef PubMed

[96] Morton GJ,

[97] Cummings DE,

[98] Baskin DG,

[99] Barsh GS,

[100] Schwartz MW

[101] ↵
Arora S,
Anubhuti
(2006) Role of neuropeptides in appetite regulation and obesity: A review. Neuropeptides 40:375–401.
OpenUrl CrossRef PubMed

[102] Arora S,

[103] Anubhuti

[104] ↵
Morrison CD,
Morton GJ,
Niswender KD,
Gelling RW,
Schwartz MW
(2005) Leptin inhibits hypothalamic NPY and AgRP gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling. Am J Physiol 289:E1051–E1057.
OpenUrl

[105] Morrison CD,

[106] Morton GJ,

[107] Niswender KD,

[108] Gelling RW,

[109] Schwartz MW

[110] ↵
Morton GJ,
et al.
(2005) Leptin regulates insulin sensitivity via phosphatidylinositol-3-OH kinase signaling in mediobasal hypothalamic neurons. Cell Metab 2:411–420.
OpenUrl CrossRef PubMed

[111] Morton GJ,

[112] et al.

[113] ↵
Ren D,
Li M,
Duan C,
Rui L
(2005) Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice. Cell Metab 2:95–104.
OpenUrl CrossRef PubMed

[114] Ren D,

[115] Li M,

[116] Duan C,

[117] Rui L

[118] ↵
Minokoshi Y,
et al.
(2002) Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339–343.
OpenUrl CrossRef PubMed

[119] Minokoshi Y,

[120] et al.

[121] ↵
Unger RH
(2004) The hyperleptinemia of obesity: Regulator of caloric surpluses. Cell 117:145–146.
OpenUrl CrossRef PubMed

[122] Unger RH

[123] ↵
Wang MY,
et al.
(2008) Adipogenic capacity and the susceptibility to type 2 diabetes and metabolic syndrome. Proc Natl Acad Sci USA 105:6139–6144.
OpenUrl Abstract/FREE Full Text

[124] Wang MY,

[125] et al.

[126] ↵
O'Gorman S,
Dagenais NA,
Qian M,
Marchuk Y
(1997) Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells. Proc Natl Acad Sci USA 94:14602–14607.
OpenUrl Abstract/FREE Full Text

[127] O'Gorman S,

[128] Dagenais NA,

[129] Qian M,

[130] Marchuk Y

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