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Leptin is an adipocyte-derived hormone that plays a key role inenergy homeostasis, yet resistance to leptin is a feature ofmost cases of obesity in humans and rodents. In vitro analysissuggested that the suppressor of cytokine signaling-3 (Socs3)is a negative-feedback regulator of leptin signaling involved inleptin resistance. To determine the functional significance ofSocs3 in vivo, we generated neural cell−specific SOCS3conditional knockout mice using the Cre–loxP system.Compared to their wild-type littermates, Socs3-deficient miceshowed enhanced leptin-induced hypothalamic Stat3 tyrosinephosphorylation as well as pro-opiomelanocortin (POMC)induction, and this resulted in a greater body weight loss andsuppression of food intake. Moreover, the Socs3-deficient micewere resistant to high fat diet−induced weight gain andhyperleptinemia, and insulin-sensitivity was retained. Thesedata indicate that Socs3 is a key regulator of diet-inducedleptin as well as insulin resistance. Our study demonstrates thenegative regulatory role of Socs3 in leptin signaling in vivo,and thus suppression of Socs3 in the brain is a potentialtherapy for leptin-resistance in obesity.

It was initially proposed that leptin has a primary role as an anti-obesity hormone; however, high concentrations of serum leptin levelsare often associated with obesity, suggesting the presence of leptinresistance1−3. The leptin receptor is highly expressed in the hypothala-mus and belongs to the cytokine-receptor superfamily that activatesthe Janus kinase−signal transducers and activators of transcription(Jak-Stat) pathway4. Stat3 is apparently essential for leptin regulationof energy balance5−7. Several mechanisms for leptin resistance havebeen documented, including impaired leptin transport across theblood-brain barrier8 and the presence of negative regulators of leptinsignaling, such as the tyrosine phosphatases SH2-containing proteintyrosine phospatase-2 (SHP-2) (ref. 9) and protein-tyrosine phos-phatase 1B (PTP1B)10−12 as well as the suppressors of cytokine signal-ing family of molecules13. Peripheral leptin administration to micerapidly induces Socs3 mRNA in hypothalamic regions14. Socs3 bindsto the phosphorylated leptin receptor through its Src homology-2

(SH2) domain and inhibits Jak tyrosine kinase activity through its N-terminal kinase inhibitory region, which functions as a pseudo-substrate13,15,16. Furthermore, the C-terminal Socs box recruits theubiquitin-transferase system and may mediate the degradation ofreceptor-Jak complexes17. Thus, Socs3 may be an important regulatorof leptin signaling and could be involved in leptin-resistance. However,because these conclusions were based on the analysis of cell lines, thephysiological significance of Socs3 in leptin signaling and its impacton obesity remained an open question.

Because Socs3-deficient mice die during embryonic development asa result of placental defects18, a conditional knockout approach wasused to determine the tissue-specific roles of Socs3 (ref. 19; Fig. 1a). Togenerate a neural cell−specific deletion, cre transgenic mice, in whichCre recombinase is expressed specifically in neural precursor cellsunder the nestin promoter-enhancer (crenes)20 or synapsin promoter(cresyn)21, were crossed with Socs3flox/flox mice. Genomic DNAextracted from the brain was analyzed with PCR to verify deletion ofSocs3. A 250−base pair band corresponding to the deleted Socs3 allele(Socs3del) appeared in the PCR analysis of brain DNA fromcrenesSocs3flox/flox (NesKO) mice and cresynSocs3flox/flox (SynKO) micebut not in the DNA from control Socs3flox/flox (wild type) mice (Fig. 1b). In these two types of conditional knockout mice, we con-firmed Socs3 deletion in the hypothalamus and cerebrum, but not inother tissues including heart, liver, kidney muscle and fat(Supplementary Fig. 1 online). RT-PCR analysis also indicated thatSocs3 expression was greatly reduced in the brain but not in the othertissues examined (Supplementary Fig. 1 online).

After leptin administration, Socs3 is exclusively expressed in thearcuate nucleus (ARC) of the hypothalamus, which coexpress as leptinreceptors and neuropeptides that are considered to be essential in theregulation of food intake14. Consistent with previous reports, our realtime RT-PCR analysis revealed that leptin treatment led to a twofoldincrease in Socs3 mRNA in wild-type mice (Fig. 1c). However, expres-sion of Socs3 mRNA in NesKO and SynKO mice was much lower thanthat in wild-type mice, irrespective of leptin administration (Fig. 1c).Immunohistological analysis with an antibody to Socs3 confirmed amarked induction of Socs3 protein in the ARC of the brain in

1Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.2Department of Molecular Genetics, Institute of Life Science, Kurume University, 2432-3 Aikawa-machi, Kurume 839-0861, Japan. 3Institute of Molecular Medicineand Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0641, USA. 4Cardiovascular Research Institute andThe Third Department of Internal Medicine, Kurume University, 67 Asahi-machi, Kurume 830-0011, Japan. Correspondence should be addressed to A. Y.(yakihiko@bioreg.kyushu-u.ac.jp).

Published online 20 June 2004; doi:10.1038/nm1071

Socs3 deficiency in the brain elevates leptin sensitivity andconfers resistance to diet-induced obesityHiroyuki Mori1, Reiko Hanada2, Toshikatsu Hanada1, Daisuke Aki1, Ryuichi Mashima1, Hitomi Nishinakamura1,Takehiro Torisu1, Kenneth R Chien3, Hideo Yasukawa4 & Akihiko Yoshimura1

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wild-type mice after the leptin injection, but only a marginal level ofinduction in the two brain-specific Socs3-deficient mice (Fig. 1d).

To clarify the effects of Socs3 deficiency on leptin-induced Stat3 acti-vation, recombinant leptin was injected into mice and the hypothala-mus was examined with immunoblotting and immunofluorescencestaining with phospho-specific (Tyr705) Stat3 antibodies. Stat3 phos-phorylation in the hypothalamus was evident within1 h, after leptininjection but decreased thereafter in wild-type mice (Fig. 2a,b).However, Stat3 activation 1 h after leptin stimulation was significantlygreater in both NesKO and SynKO mice com-pared to their control littermates, and phos-phorylation was sustained for much longer.The ARC of the hypothalamus showed anincrease in both the number and intensity ofneurons positively staining with phospho-

Stat3 (Fig. 2c). Prolonged activation of Stat3in knockout mice was confirmed withimmunofluorescence microscopy (Fig. 2c). Astudy that used mice carrying a mutant leptinreceptor lacking the Stat3 binding siterevealed that Stat3 plays a major role in stim-ulating the catabolic pathways by inducingexpression of the pro-opiomelanocortin(POMC) product α-MSH6,22. The level of

POMC induced by leptin administration was greater in Socs3-deficient mice than in wild-type mice (Fig. 2d). These data indicatethat Socs3 is a negative regulator of leptin-induced Stat3 signaling inthe hypothalamus.

We then examined the effect of Socs3 deletion on body weight lossinduced by leptin. Although leptin at a dose of 1.5 µg/g body weightended to cause a slight decrease in body weight in wild-type mice, thesame dose of leptin infused into either of the two types of neuron-specific Socs3-deficient mice resulted in greater weight loss (Fig. 3a).

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Food intake was also suppressed more in NesKO and SynKO mice thanin control wild-type mice (Fig. 3b). Similar effects of Socs3 deletion onbody weight loss and food intake were observed in both male andfemale mice. Pair-feeding experiments indicated that the leptin-induced body weight loss observed in knockout mice was due to areduced food intake (data not shown). Greater body weight loss inSocs3-deficient mice than in wild-type mice was observed throughouta range of leptin doses (0.5−4.5 µg/g body weight, Supplementary Fig.2 online). Circulating leptin levels after leptin injection did not differbetween wild-type and knockout mice (Supplementary Fig. 3 online).These data suggested that Socs3 deletion in the brain resulted in anenhanced leptin signaling, which led to a greater suppression of foodintake and body weight loss.

To examine the role of Socs3 in obesity-associated leptin resistance,mice were placed on a high fat diet (HFD). During 22 weeks on theHFD, both male and female wild-type mice rapidly gained weight (Fig. 4a). However, Socs3-deficient mice on the HFD gained signifi-cantly less weight compared to their wild-type littermates (Fig. 4a,b).Although the snout-anus length did not significantly differ (data notshown), the weight of each fat pad in Socs3-deficient mice on the HFDwas significantly lower than that of wild-type mice (Fig. 4c). Socs3-deficient mice also ingested significantly less food than their wild-typelittermates (Fig. 4d). As expected, after 22 weeks on the HFD, plasmatriglyceride and free-fatty acid levels were significantly lower in condi-tional knockout mice than in wild-type mice (Supplementary Fig. 4online). Plasma leptin levels in wild-type mice on the HFD were

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∼ 5 ng/ml, suggesting the development of hyperleptinemia and leptinresistance, whereas Socs3-deficient mice showed lower leptin levels,which were similar to the levels seen under normal diet conditions(Fig. 4e). These data show that wild-type mice on the HFD developedobesity and leptin resistance. In contrast, Socs3-deficient mice on theHFD showed a reduced appetite and body weight gain and leptinresistance was rescued in these mice.

Leptin can improve insulin resistance and diabetes in a mousemodel of lipoatrophic diabetes23 and so we examined insulin sensitiv-ity in 28- to 30-week-old NesKO mice and control littermates on anHFD using intraperitoneal glucose- and insulin-tolerance tests. Abolus of glucose was more rapidly cleared by NesKO mice than controlmice (Fig. 4f). Hyperglycemia was more pronounced in the wild-typeanimals than in Socs3-deficient mice at all time points after glucoseadministration. The insulin tolerance test revealed that wild-type miceon the HFD were insulin-resistant, whereas Socs3-deficient mice weresensitive to insulin (Fig. 4g). Taken together, these results demonstratethat ablation of Socs3 expression in neurons ameliorates HFD-induced insulin resistance.

The improvement of glucose tolerance and insulin sensitivity maybe explained by the reduced adiposity in Socs3 knockout mice.However, adiposity is also regulated by brain insulin24, and insulinreceptor gene deletion in the brain resulted in increased food intakeand diet-induced obesity25. Socs3 inhibits insulin signaling by induc-ing the degradation of IRS-1 and IRS-2 (ref. 26) or by direct binding tothe insulin receptor27. Therefore, one can not rule out the possibilitythat Socs3 inhibits insulin signaling in the brain and that this influ-ences propensity to diet-induced obesity and peripheral insulin resist-ance. Further study is necessary to address this point.

Our study suggests that excessive energy intake and body fat deposi-tion in response to a HFD leads to the acquisition of resistance to bothleptin and insulin, whereas loss of Socs3 expression in the brain ame-liorates diet-induced obesity, leptin resistance and even insulin resist-ance. This raises the possibility that the Stat3-Socs3 system in the brainplays a major role in regulating the amount of body fat. This work alsomakes brain Socs3 a potential therapeutic target for the treatment ofleptin resistance, type 2 diabetes and obesity.

METHODSGeneration of conditional Socs3-deficient mice. Socs3flox/flox mice were asdescribed19,28. Nestin-Cre mice expressing Cre recombinase under the controlof the mouse nestin gene promoter (a gift from H. Okano, Keio University,Kanagawa, Japan and S. Noguchi, Meiji Institute of Health Science, Meiji MilkProducts) were as described20,26 . Synapsin1-Cre mice21 were from The JacksonLaboratory. Nestin-Cre mice and Synapsin1-Cre mice were crossed toSocs3flox/flox mice to generate offspring carrying both the cre gene and the heterozygous loxP-flanked Socs3 mutation (Nes-creSocs3flox/+ or Syn-creSocs3flox/+). These mice were further mated with Socs3flox/flox mice, and Nes-creSocs3flox/flox or Syn-creSocs3flox/flox mice were generated. Socs3flox/flox

mice were generated as 129/Bl6 mixed background, and then backcrossed threetimes into C57Bl/6. Nestin-Cre and synapsin-Cre transgenic mice were back-crossed into C57Bl/6 mice at least five times. All animals used in this study weregenerated by crossing creSocs3flox/flox (knockout) and Socs3flox/flox (wild-type)mice and we used littermates for experiments. Genotyping was done withprimers as described19,27 (Fig. 1a). Mice were housed in a 12 h light:dark cycleand temperature-controlled environment. Animal care and all experimentswere conducted in accordance with the institutional guidelines of the KyushuUniversity Graduate School of Medicine.

HFD and leptin administration. Male and female wild-type and Socs3 knock-out mice were fed on a normal diet (low fat: 6% calories from fat). Then 8-week-old NesKO or SynKO and wild-type mice were placed on a HFD com-posed of 23% calories from fat (Oriental Kobo) for up to 30 weeks. For a leptin

administration study, mice from 18 to 26 weeks of age were individually caged.Murine leptin (PeproTech) (0.5−4.5 µg/g body weight) was injected intraperi-toneally twice daily (at 08:30 and at 19:30) for 3 days. Body weight and foodintake were measured daily from 08:00 to 08:30.

Detection of hypothalamic Stat3 and Socs3. Murine leptin (3.0 µg/g bodyweight) or PBS was administered intraperitoneally to mice that had fasted for18 h. After injection, the mice were killed, at 08:30−10:30: the hypothalamuswas isolated and lysed, as described6. Lysates were resolved with SDS-PAGE andmembranes were immunoblotted with anti−phospho-Stat3 (Tyr705) and anti-Stat3 (Cell Signaling). A macrophage cell line (RAW) stimulated with LPS for 3 h in vitro was used as positive control. For quantitative analysis, density of theblots for phosphorylated Stat3 and total Stat3 was measured and expressed asratios of phosphorylated Stat3 to total Stat3. For phospho-Stat3 and Socs3immunofluorescence detection, male wild-type and NesKO mice were injectedintraperitoneally with 3.0 µg/g body weight of leptin and perfused withformaldehyde 3 h after leptin treatment. Hypothalamic sections from micewere also immunostained with anti-Socs3 (IBL) or anti-phospho-Stat3 using areported protocol in rats29.

RNA extraction and RT-PCR. Socs3 knockout and control mice were fasted for18 h to suppress leptin levels and then injected with 3.0 µg/g body weight ofleptin. The hypothalamus was isolated from 08:30 to 10:30 and total RNA waspurified, as described6. We quantified the expression of POMC and Socs3mRNA using RT-PCR as described6,28.

Metabolic measurements. Twenty-eight-week-old male mice fed on a HFD for22 weeks were fasted overnight, and blood was collected from the retro-orbitalsinus at 09:00. Blood glucose was measured with an Antsense-II glucometer(Sankyo). The serum leptin concentration was determined with enzyme-linkedimmunosorbent assay (ELISA) kits (Morinaga).

Glucose and insulin tolerance tests. Male mice were used because they are morehormonally stable. For a glucose tolerance test, 28-week-old NesKO mice or lit-termates fed on a HFD were fasted overnight and glucose was administeredintraperitoneally (1.5 mg/g body weight). For the insulin tolerance test,30-week-old male mice were fed ad libidum, and 0.5 mU/g body weight ofhuman insulin (Novolin R, Novo Nordisk) was injected intraperitoneally. Theglucose levels were measured in blood withdrawn from the tail.

Statistical analysis. Data are expressed as the mean ± s.e.m. Statistical signifi-cance was tested with an unpaired two-tailed Student’s t-test and analysis ofvariance (ANOVA). The differences were considered to be significant ifP < 0.05.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTSWe thank Y. Kawabata for technical assistance; N. Arifuku, F. Yamaura and Y. Nishifor manuscript preparation; P. Kievit and J.S. Flier for discussions; and M. Oharafor language assistance. Supported by special grants-in-aid from the Ministry ofEducation, Science, Technology, Sports, and Culture of Japan, the Japan HealthScience Foundation, Mochida Memorial Foundation, and the Uehara MemorialFoundation.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 11 March; accepted 2 June 2004Published online at http://www.nature.com/naturemedicine/

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