Hepatic Oncostatin M Receptor β Regulates Obesity-Induced Steatosis and Insulin Resistance

Hepatic Oncostatin M Receptor β Regulates Obesity-Induced Steatosis and Insulin Resistance

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The American Journal of Pathology, Vol. 186, No. 5, May 2016

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METABOLIC, ENDOCRINE, AND GENITOURINARY PATHOBIOLOGY

Hepatic Oncostatin M Receptor b Regulates Obesity-Induced Steatosis and Insulin Resistance Q35

Q1

Pengcheng Luo,*y Pi-Xiao Wang,zx Zuo-Zhi Li,{ Xiao-Jing Zhang,k Xi Jiang,zx Jun Gong,zx** Juan-juan Qin,zx Junhong Guo,zx Xueyong Zhu,zx Sijun Yang,x and Hongliang Lizx From the Departments of Nephrology* and Cardiology,z Renmin Hospital of Wuhan University, Wuhan; the Huangshi Central Hospital,y Hubei Polytechnic University, Huangshi, Hubei Province; the Animal Experiment Center/Animal Biosafety Level-III Laboratory,x and the College of Life Sciences,** Wuhan University, Wuhan; the Department of Cardiology,{ Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing; and the State Key Laboratory of Quality Research in Chinese Medicine,k Institute of Chinese Medical Sciences, University of Macau, Macao, China Accepted for publication December 31, 2015. Address correspondence to Hongliang Li, M.D., Ph.D., Department of Cardiology, Renmin Hospital of Wuhan University, Animal Experiment Center/Animal Biosafety LevelIII Laboratory, Collaborative Innovation Center of Model Animal, Cardiovascular Research Institute, Wuhan University, Luojia Mount Wuchang, Wuhan 430072, China. E-mail: [email protected].

Q4

The liver is an essential insulin-responsive organ that is critical for maintaining glucose homeostasis and lipid metabolism. Oncostatin M receptor b chain (OSMRb) is implicated in adipose tissue- and immune cell-mediated metabolic regulation. However, the role of hepatocyte-derived OSMRb in metabolic disorders remains unclear. Here, we report on the central role of OSMRb in the protection against obesity and deregulation of glucose and lipids. We observed significantly varied expression levels of OSMRb in hepatic tissues in both human samples and mouse models of nonalcoholic fatty liver disease. Mice lacking either whole-body or hepatic OSMRb displayed exacerbated diet-induced insulin resistance, hepatic steatosis, and inflammation, both in diet-induced and genetically (ob/ob) obese mice. These adverse effects were markedly attenuated by hepatocyte-specific overexpression of OSMRb. Mechanistically, we showed that OSMRb phosphorylates and activates the Janus kinase 2 (JAK2)/STAT3 signaling pathway in the liver. More importantly, the liver-restricted overexpression of STAT3 rescued glucose tolerance and ameliorated hepatic steatosis and inflammation in OSMRb knockout mice, whereas OSMRb overexpression failed to protect against hepatic steatosis, insulin resistance, and hepatic inflammation in STAT3-deficient mice. Thus, activation of STAT3 is both sufficient and required to produce OSMRb-mediated beneficial effects. In conclusion, hepatic OSMRb expression alleviates obesity-induced hepatic insulin resistance and steatosis through the activation of JAK2/STAT3 signaling cascades. (Am J Pathol 2016, 186: 1e15; http://dx.doi.org/10.1016/j.ajpath.2015.12.028)

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disorder in the Western world and affects up to 30% of the general population and 75% to 100% of obese individuals.1,2 Most patients with NAFLD manifest a certain degree of hepatic insulin resistance (IR) and dyslipidemia,1 with obesity closely linked to the development of insulin resistance and diabetes mellitus.3 The ectopic accumulation of hepatic lipids is an early manifestation of NAFLD that inhibits the insulin receptor kinase,2 and increased serum concentrations of glucose and lipids may increase the affected individual’s susceptibility to oxidative damage and inflammation.4 Insulin regulation involves an intricate relay of signaling cascades that include the phosphorylation and activation of insulin receptor substrates (IRSs) and downstream AKT and Forkhead box protein O1 (FOXO1).5,6 Defects in insulin-induced signaling

cascades coupled with nutritional oversupply contribute to the deregulation of lipid and glucose metabolism. Unfortunately, current therapies for NAFLD are limited to weight loss, and effective drug therapies have not been developed.5 Thus, novel therapies for NAFLD are warranted. Supported by the National Science Fund for Distinguished Young Q2 Scholars grant 81425005 (H.L.); the National Natural Science Foundation of China grants 81170086 and 81270184 (H.L.); National Science and Technology Support Project grants 2011BAI15B02, 2012BAI39B05, 2013YQ030923-05, 2014BAI02B01, and 2015BAI08B01 (H.L.); the Key Project of the National Natural Science Foundation grant 81330005 (H.L.); the National Basic Research Program China grant 2011CB503902 (H.L.), and Natural Science Foundation of Hubei Province grant 2013CFB259 (H.L.). Q3 P.L., P.-X.W., and Z.-Z.L. contributed equally to this work. Disclosures: None declared.

Copyright ª 2016 Published by Elsevier Inc. on behalf of the American Society for Investigative Pathology. http://dx.doi.org/10.1016/j.ajpath.2015.12.028

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The liver is an essential metabolic organ for glucose homeostasis and lipid metabolism, including lipogenesis, lipid uptake, and fatty acid b-oxidation. In the development of IR, growing evidence indicates the crucial role of hepatocytes instead of changes in circulating adipocytokines. For instance, our group and others have shown that mice presenting targeted overexpression of IRF3, IRF9, and STAT3 in the liver display ameliorated hepatic IR and liver-specific steatosis.7e9 Nevertheless, despite the increased prevalence and potentially severe consequences, the underlying mechanism of IR and hepatic steatosis are not completely understood. Oncostatin M (OSM) was initially characterized from its antiproliferative effect on A375 melanoma and is a member of the gp130 [or IL-6/leukemia inhibitory factor receptor (LIFR)] cytokine family.10 The pathophysiologic functions of OSM in the liver are diverse. OSM was initially identified as a potent inducer of hepatocyte acute-phase protein responses.11 Studies have shown that OSM is also involved in iron metabolism regulation by transcriptional induction of hepatic hepcidin.12 Moreover, OSM may also help coordinate the development of liver differentiation and hematopoiesis.13,14 Notably, OSM engages a second receptor, either LIFRa or OSM receptor b chain (OSMRb), before recruiting gp130.10 OSM/gp130 then initiates the activation of several transcription factors, including STAT3 and mitogen-activated protein kinase (MAPK), both of which are modulators of glucose regulation.9,15,16 Interestingly, OSM has been shown to up-regulate HepG2 low-density lipoprotein receptor expression, indicating a potential role in hepatic lipid metabolism, which is supported by the metabolic effect of OSM in Kupffer cells.10,17,18 Nevertheless, previous studies primarily focused on the role of OSM/ OSMRb in adipocyte and liver immune cells, and the underlying functional role of hepatocyte-derived OSMRb in the myriad interrelated disorders, including IR, dyslipidemia, hepatic inflammation, and steatosis, is unknown.17e20 Here, we found that NAFLD correlated with decreased expression levels of OSMRb in hepatic tissues in both human and mouse models. More importantly, we showed that hepatocyte-derived OSMRb provides protection against obesity-induced hepatic IR, inflammatory responses, and steatosis through the activation of Janus kinase 2 (JAK2)/ STAT3 in the liver. Furthermore, by genetically manipulating STAT3 in mice, we showed that STAT3 is both sufficient and required for OSMRb-mediated beneficial effects. Thus, targeting OSM/OSMRb may improve hepatic lipid metabolism and insulin sensitivity.

(Cre) transgenic mice [albumin (Alb)-Cre; stock no. 003574] Q7 and STAT3 conditional (floxed) mutant mice (STAT3flox/flox; stock no. 016923) were purchased from The Jackson Laboratory (Bar Harbor, ME). Full-length mouse OSMRb cDNA was inserted after a pCAG-CAT promoter that expressed the CAT gene, which was flanked by loxP sites. The pCAG-CATOSMRb construct was microinjected into fertilized embryos (C57BL/6J background) to produce OSMRb-floxed mice. Subsequently, liver-specific OSMRb transgenic (OSMRb-TG) mice were generated by crossing OSMRb-floxed mice with Alb-Cre mice, and liver-specific STAT3-knockout (KO) mice were produced by mating STAT3flox/flox mice with Alb-Cre mice. The liver-specific STAT3-TG mice were generated with similar procedures to those used to generate the OSMRbTG mice, and the primers used for genotyping were as follows: CAG forward, 50 -CCCCCTGAACCTGAAACATA-30 ; and Stat3C reverse, 50 -GCAATCTCCATTGGCTTCTC-30 . Global OSMRb-KO mice were established with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, as previously described.21 Briefly, guide sequences of the target site for OSMRb gene in the mouse genome were predicted by the online CRISPR design tool (http://crispr.mit. edu; last accessed December 3, 2015). After in vitro transcription, mouse zygotes were injected with a mixture of NLSflag-linker-Cas9 mRNA (no. 44758; Addgene, Cambridge, MA) and OSMRb-single guide RNA. DNA sequences for Q8 genotyping primers are as follows: forward, 50 -ATTGCCCAGCAAGTTCTTTG-30 , and reverse, 50 -CACACAGGGATGCAATTGTT-30 . OSMRb-KO mice were crossed with STAT3-TG mice to obtain OSMRb-KO/STAT3-TG (OKST) mice. OSMRb-TG/STAT3-KO (OTSK) mice were then produced by crossing OSMRb-TG mice with STAT3-KO mice. The ob/ob mice were purchased from The Jackson Laboratory (stock no. 000632). Eight-week-old male mice were used in this study, and they were fed either normal chow (protein, 18.3%; fat, 10.2%; carbohydrates, 71.5%; D12450B; Research Diets, Inc., New Brunswick, NJ) or a high-fat diet (HFD; protein, 18.1%; fat, 61.6%; carbohydrates, 20.3%; Q9 D12492; Research Diets, Inc.) ad libitum for up to 24 weeks. All of the animals were housed in an environment under a 12-hour light/dark cycle at a fixed temperature and humidity and given ad libitum access to food and water. Mouse body weights and fasting blood glucose concentrations were monitored every 4 weeks, and the food intake amounts were monitored weekly. The experimenters (X.-J.Z., X.J., J.Go., J.-j.Q., J.Gu., and X.Z.) were blinded Q10 to the genotypes of the animals.

Tissue Preparation

Materials and Methods Q6

Animals and Diets All of the animal experiments were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. Liver-specific cyclization recombination enzyme

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After anesthetization with 3% pentobarbital sodium, the animals were euthanized, and the livers were immediately removed and weighed. Each liver was divided into two parts, with one part immediately frozen in liquid nitrogen at 80 C and the other part fixed in 10% formalin or frozen with Tissue-Tek OCT Compound (Tokyo, Japan) in dry ice

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OSMRb Attenuates Hepatic Steatosis 311 312 313 314 315 316 Assessment of Liver Function 317 Metabolic Studies and Serum Cytokine Analyses 318 The concentrations of alanine transaminase and aspartate 319 For the glucose tolerance tests (GTTs) and insulin tolerance transaminase in the serum were measured with an ADVIA 320 tests (ITTs), 1 g/kg glucose (Sigma-Aldrich Co., St. Louis, 2400 Chemistry System analyzer (Siemens, Tarrytown, NY) 321 MO) or 0.75 U/kg insulin (Novolin R; Novo Nordisk Co., 322 according to the manufacturer’s instructions. 323 Bagsvaerd, Denmark), respectively, were intraperitoneally 324 injected into mice. Blood glucose concentrations were detecQuantitative Real-Time PCR 325 ted with a glucometer (One Touch Ultra Easy; LifeScan, Inc., 326 Milpitas, CA) after a 6-hour fast before injection and 15, 30, Total RNA was extracted from liver tissue with the use of 327 60, and 120 minutes after glucose or insulin injection. Serum TRIzol reagent (Invitrogen) according to the manufacturer’s 328 fasting insulin was examined by enzyme-linked immunosorprotocol and was reverse-transcribed into cDNA with the 329 bent assay (Millipore, Billerica, MA). The homeostasis model use of a Transcriptor First-Strand cDNA Synthesis Kit 330 assessment of the IR (HOMA-IR) index was calculated as (Roche, Indianapolis, IN). A LightCycler 480 SYBR Green 331 HOMA-IR Z [FBG (mmol/L)  FINS (mIU/L)]/22.5. 1 Master Mix (Roche) and LightCycler 480 QPCR System 332 Hepatic triglyceride, total cholesterol (TC), and nones(Roche) were used to perform the quantitative real-time 333 PCR analysis. The PCR conditions were as follows: 95 C terified fatty acid (NEFA) concentrations were determined 334 335 with commercial kits (Wako Chemicals, Richmond VA). for 10 minutes; 40 cycles of 95 C for 10 seconds, 60 C for 336 b-Hydroxybutyrate serum concentrations were examined 10 seconds, and 72 C for 20 seconds; and a final extension 337 with commercial kits (Abcam, Cambridge, UK). Serum at 72 C for 10 minutes. The relative quantity of the mRNAs 338 cytokines IL-1b, IL-6, IL-4, tumor necrosis factor (TNF)-a, was calculated by normalization to the quantity of GAPDH 339 monocyte chemoattractant protein-1 (MCP1), IL-10, leptin, mRNA. The primer pairs are listed in Table 1. ½T1 340 341 Q34 342 Table 1 Primers for Real-Time PCR Detection 343 Gene Forward primer Reverse primer 344 50 -TCTCCATGGTGGTGAAGACA-30 GAPDH 50 -ACTCCACTCACGGCAAATTC-30 345 OSMRB 50 -TTTGTGCGCTGTGCAAGTGC-30 50 -TGCGTCTTCCATTCTCCGACC-30 346 G6PC 50 -TCTGTCCCGGATCTACCTTG-30 50 -GCTGGCAAAGGGTGTAGTGT-30 347 ABCG1 50 -TGAACCCGTTTCTTTGGCACCG-30 50 -AGTCCCGCATGATGCTGAGGAA-30 348 CYP7A1 50 -TCAAAGAGCGCTGTCTGGGTCA-30 50 -TTTCCCGGGCTTTATGTGCGGT-30 349 SREBP1c 50 -CACTTCTGGAGACATCGCAAAC-30 50 -ATGGTAGACAACAGCCGCATC-30 350 HMGCR 50 -ATCATGTGCTGCTTCGGCTGCAT-30 50 -AAATTGGACGACCCTCACGGCT-30 351 CD36 50 -TGGGTTTTGCACATCAAAGA-30 50 -GATGGACCTGCAAATGTCAGA-30 352 FABP1 50 -TGGTCCGCAATGAGTTCACCCT-30 50 -CCAGCTTGACGACTGCCTTGACTT-30 353 FATP1 50 -TGCACAGCAGGTACTACCGCAT-30 50 -TGCGCAGTACCACCGTCAAC-30 354 PPARG 50 -ATTCTGGCCCACCAACTTCGG-30 50 -TGGAAGCCTGATGCTTTATCCCCA-30 355 ACCA 50 -GGCCAGTGCTATGCTGAGAT-30 50 -AGGGTCAAGTGCTGCTCCA-30 356 SCD1 50 -TCTTCCTTATCATTGCCAACACCA-30 50 -GCGTTGAGCACCAGAGTGTATCG-30 357 FAS 50 -CTGCGGAAACTTCAGGAAATG-30 50 -GGTTCGGAATGCTATCCAGG-30 358 PDK4 50 -TTCACACCTTCACCACATGC-30 50 -AAAGGGCGGTTTTCTTGATG-30 359 UCP2 50 -GCTGGTGGTGGTCGGAGATA-30 50 -ACTGGCCCAAGGCAGAGTT-30 360 MCAD 50 -TGGCGTATGGGTGTACAGGG-30 50 -CCAAATACTTCTTTTTTTGTTGATCA-30 361 LCAD 50 -GGAGTAAGAACGAACGCCAA-30 50 -GCCACGACGATCACGAGAT-30 362 ACOX1 50 -CGGAAGATACATAAAGGAGACC-30 50 -AAGTAGGACACCATACCACCC-30 363 CPT1A 50 -AGGACCCTGAGGCATCTATT-30 50 -ATGACCTCCTGGCATTCTCC-30 364 PPARA 50 -TATTCGGCTGAAGCTGGTGTAC-30 50 -CTGGCATTTGTTCCGGTTCT-30 365 IL1B 50 -CCGTGGACCTTCCAGGATGA-30 50 -GGGAACGTCACACACCAGCA-30 366 IL6 50 -AGTTGCCTTCTTGGGACTGA-30 50 -TCCACGATTTCCCAGAGAAC-30 367 TNFA 50 -CATCTTCTCAAAATTCGAGTGACAA-30 50 -TGGGAGTAGACAAGGTACAACCC-30 368 MCP1 50 -TAAAAACCTGGATCGGAACCAAA-30 50 -GCATTAGCTTCAGATTTACGGGT-30 369 iNOS 50 -TGCGCCTTTGCTCATGACATCGA-30 50 -ATGGATGCTGCTGAGGGCTCTGTT-30 370 IL10 50 -CCAAGCCTTATCGGAAATGA-30 50 -TTTTCACAGGGGAGAAATCG-30 371 372

before embedding. Liver tissues sections were prepared and stained with hematoxylin and eosin (5 mm per section) or Oil Red O stain (4 mm). The sections were then counterstained with Mayer hematoxylin after destaining in 60% isopropanol.

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resistin, and adiponectin were assessed via enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN; MBL International, Woburn, MA; RayBiotech, Norcross, GA; Invitrogen, Carlsbad, CA; PeproTech, Rocky Hill, NJ).

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Western Blot Analysis Antibodies against OSMRb (sc30010) were purchased from Santa Cruz Biotechnology (Dallas, TX). The antibody against p-IRS1 (09-432) was purchased from Millipore (Billerica, MA). Antibodies to IRS1 (2382), p-Akt (4060), Akt (4691), p-GSK3b (9322), GSK3b (9315), p-FOXO1 (9461), FOXO1 (2880), IKKb (2370), p-P65 (3033), P65 (4764), p-IkBa (9246), IkBa (4814), p-MEK1/2 (9154), MEK1/2 (9122), p-ERK1/2 (4370), ERK1/2 (4695), p-P38 (4511), P38 (9212), p-JNK (4668), JNK (9258), p-JAK2 (3776), JAK2 (3230), p-STAT3 (9145), and GAPDH (2118) were obtained from Cell Signaling Technology (Boston, MA). The STAT3 antibody (BS1336) was obtained from Bioworld Technology (Harrogate, UK), and antibody against p-IKKb (Ab5915) was ordered from Abcam. The livers or cultured cells were lyzed in lysis buffer, and 50 mg of extracted protein was separated on 8% to 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes Q13 were blocked in tris-buffered saline and Tween 20 that contained 5% skim milk powder for 1 hour at room temperature and then incubated in primary antibodies at 4 C overnight. Next, the membranes were washed and then incubated with secondary antibodies for 1 hour at room temperature. A ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA) was used for signal detection. Protein Q14 expression levels were quantified with Image Lab Software version 5.1 (Bio-Rad) and normalized to the loading control GAPDH.

Cell Culture and in Vitro Model of Hepatic Steatosis

Q15

After anesthetization, mouse livers were perfused with perfusion buffer (0.9% saline) in situ through the superior vena cava, followed by digestion buffer (D Hanks’ solution supplemented with 0.05% trypsin 25200; Gibco, Carlsbad, CA). The cell suspension was filtered and then centrifuged at 50  g for 5 minutes. The harvested hepatocytes were then resuspended in Dulbecco’s modified Eagle’s medium (15% fetal bovine serum, 5 mg/mL insulin, 100 U/mL penicillin, 100 U/mL streptomycin) before seeding on collagen-coated dishes (Sigma-Aldrich Co.). After cultivation for 24 hours (5% CO2, 37 C), the medium was replaced with fresh fetal bovine serum-free media, and the cells were then incubated for another 24 hours before adenovirus administration. To establish an in vitro model of hepatic steatosis,22 palmitate (0.25 mmol/L; Sigma-Aldrich Co.) was added to the medium of cultured hepatocytes and incubated for an additional 24 hours.

Recombinant Adenovirus Vector Production and Transfection Adenoviruses carrying sequences encoding mouse OSMRb (AdOSMRb) and a shRNA targeting OSMRb (AdshOSMRb)

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were constructed as previously described.23 Similar adenoviral vectors encoding the GFP gene (AdGFP) or scrambled shRNA (AdshRNA) were used as controls. The adenoviruses were transfected into cultured hepatocytes at a multiplicity of infection of 100 for 48 hours. For in vivo studies, we injected appropriate adenoviruses (5  109 plaque-forming units) into the jugular vein as previously described.23 Four weeks after adenoviral injection, the mice were euthanized, and their tissues were harvested.

Human Liver Tissue Samples Samples of human NAFLD were collected from NAFLD patients undergoing liver biopsy to diagnose fatty liver. Control samples were obtained from normal liver donors who died from accidents, but their livers were not suitable for transplantation for nonhepatic reasons. Clinical and histologic characteristics of these samples are provided in Supplemental Tables S1 and S2. Written informed consent was obtained from the families of the prospective liver donors. All procedures that involved human samples were approved by the Renmin Hospital of Wuhan University Review Board, Wuhan, China.

Statistical Analysis All data are expressed as the means  SD. Differences among groups were determined with a one-way analysis of variance with a subsequent least squares difference test (assuming equal variances) or Tamhane’s T2 test (without the assumption of equal variances). Comparisons between two groups were performed with the two-tailed Student’s t-test. P < 0.05 was considered significant. The data analysis and imaging studies were performed in a blinded Q16 manner (X.-J.Z., X.J., J.Go., J.-j.Q., and J.Gu.).

Results OSMRb Protein Expression Is Decreased in Mice with Diet-Induced and Genetic Obesity To determine the involvement of OSMRb in humanpathologic processes, we first investigated OSMRb expression levels in the liver samples of patients with NAFLD. Interestingly, OSMRb expression in the NAFLD livers was reduced to approximately 35.7% that of the normal controls (Figure 1A). OSMRb protein expression ½F1 was also reduced in a cell model of steatosis induced by palmitate treatment (Figure 1B).22 Next, we examined hepatic OSMRb expression in HFD-induced and genetic (ob/ob) obesity models, both of which induce several features of human NAFLD, including lipid accumulation, inflammation, and hepatic steatosis.24 Mice were fed a normal chow diet or HFD for 24 weeks. The HFD administration for 8 weeks led to significantly up-regulated protein expression of OSMRb, whereas hepatic OSMRb protein

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OSMRb Attenuates Hepatic Steatosis

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2 0 WT/HFD 0 wks WT/HFD 16 wks

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Figure 1 OSMRb protein expression is reduced in the livers of obese mice and NAFLD patients. A: Total liver homogenates were harvested from human control or NAFLD samples and subjected to immunoblotting. B: Primary hepatocytes were challenged with 0.25 mmol/L palmitate, and protein levels of OSMRb was detected via immunoblotting. C and D: Mouse liver homogenates were obtained at the indicated times. Protein expression and quantification of OSMRb. AeD: GAPDH was used as a loading control. E and F: The mRNA expressions of OSMRb in the liver of WT mice fed an HFD (E) or ob/ob mice and lean controls (F). Data are expressed as means  SD. n Z 4 for normal and n Z 6 for NAFLD (A); n Z 4 independent experiments (B); n Z 4 per time point (C and D). *P < 0.05 versus control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HFD, high-fat diet; NAFLD, nonalcoholic fatty liver disease; OSMRb, OSM receptor b chain; WT, wild-type.

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was dramatically down-regulated in mice challenged with HFD for 16 or 24 weeks compared with that of the control group (Figure 1C). Interestingly, mRNA levels of hepatic OSMRb remained at a higher level between 8 and 24 weeks after HFD administration than for NC controls (Figure 1E). Moreover, a similar expression profile of hepatic OSMRb expression was observed in the ob/ob mice (Figure 1, D and F). Collectively, this reduction in hepatic OSMRb protein expression indicates potential biological significance in both mice and humans.

OSMRb Deletion Exacerbates Obesity and Metabolic Dysfunction Because hepatic OSMRb was observed to decrease in obese mice and NAFLD patients, we next investigated the biological effect of this reduction on metabolic disorders. To address this issue, global OSMRb-KO mice were established with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system (Supplemental Figure S1, AeC), and OSMRb ablation was confirmed with direct sequencing of PCR products (Supplemental Figure S1D) and

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ob/ob 8 wks ob/ob 24 wks

immunoblotting (Supplemental Figure S1E). Intriguingly, although body weight was comparable between OSMRb-KO and wild-type (WT) mice fed an NC diet, HFD-induced obesity was more severe in the OSMRb-KO mice than in their WT littermates (Figure 2A). Functionally, glucose metabolic disorders were also exacerbated on OSMRb ablation as evidenced by significantly higher fasting blood glucose (Figure 2B) and insulin concentrations (Figure 2C) in the OSMRb-KO mice than in the WT controls. In addition, OSMRb-KO mice also exhibited a higher HOMA-IR index than that of the WT mice (Figure 2D). We then performed i.p. GTTs and i.p. ITTs in which OSMRbKO mice exhibited defective insulin sensitivity compared with WT mice (Figure 2, E and F). In response to insulin stimulation, IRS1 is recruited to the activated insulin receptor, which in turn phosphorylates and activates IRS1; the subsequent AKT/GSK3b/FOXO1 phosphorylation cascade plays a central role in metabolic homeostasis. Immunoblotting showed that insulin-induced phosphorylation of IRS1, AKT, GSK3b, and FOXO1 in OSMRb-KO mice was decreased compared with that of the WT controls, indicating impaired insulin regulation

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(Figure 2G). Considering that the global OSMRb-KO mice were used, we next examined its impacts on AKT activation in adipose tissue and skeletal muscle. On insulin stimulation, OSMRb-KO mice displayed significantly reduced expression level of phosphorylated AKT compared with that of WT mice in both adipose tissue and skeletal muscle (Supplemental Figure S2A). Under pathophysiologic conditions, hepatic OSMRb expression levels were reduced after HFD-induced obesity (Figure 1B); therefore, we next determined whether a liverspecific reduction of OSMRb expression exerted a similar deleterious effect on IR. To this end, we used adenovirusmediated gene transfer, a well-established method that acutely delivers the gene of interest to the liver by jugular injection.7,8 Injections of adenoviruses carrying sequences encoding a shRNA targeting OSMRb (AdshOSMRb) were administered to mice fed an HFD for 20 weeks. Four weeks later, OSMRb expression diminished to approximately 34.2% that of adenoviral vectors encoding scrambled shRNA

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Figure 2 Deletion of OSMRb aggravates obesity and insulin resistance. A: Body weight analysis of the WT and OSMRb-KO mice on the 24-week NC diet or HFD. B and C: Fasting serum glucose (B) and insulin (C) concentrations were assessed at the indicated time points. D: HOMA-IR index was calculated as [FBG (mmol/L)  FINS (mIU/L)]/22.5. E and F: OSMRb-KO mice display aggravated insulin resistance, and IPGTT (1 g/kg body weight) (E) and IPITT (0.75 U/kg body weight) (F) were performed on the WT and KO mice in both the NC and HFD groups. The corresponding AUC of the blood glucose concentrations in each group was calculated. G: Mouse liver homogenates were obtained from mice fed a HFD for 24 weeks, deprived of food overnight, and intraperitoneally injected with 0.75 U insulin/kg body weight for 10 minutes. Expression and quantification of the indicated proteins. Data are expressed as means  SD. n Z 14 to 21 per group (A); n Z 10 to 12 per group (B and C); n Z 10 to 12 per group at each time point (D); n Z 10 to 14 per group at each time point (E and F); n Z 3 independent experiments (G). GAPDH was used as a loading control. *P < 0.05 versus the WT NC mice; y P < 0.05 versus WT HFD mice. AUC, area under the curve; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HFD, high-fat diet; HOMA-IR, homeostasis model assessment of insulin resistance; IPGTT, i.p. glucose tolerance test; IPITT, i.p. insulin tolerance test; KO, knockout; NC, normal chow; OSMRb, OSM receptor b chain; WT, wildtype.

(AdshRNA) controls in hepatic tissues (Supplemental Figure S3A). Although the body weight was comparable (Supplemental Figure S3B), this reduction in OSMRb expression levels resulted in significantly elevated fasting serum glucose and insulin concentrations compared with that of the control animals (Supplemental Figure S3C). The i.p. GTT and ITT results indicated IR was further exacerbated in AdshOSMRb-treated mice (Supplemental Figure S3, D and E). Overall, these data show that OSMRb ablation exacerbates HFD-induced IR.

Hepatic OSMRb Overexpression Improves IR Subsequently, we proposed that if the adverse reduction of hepatic OSMRb expression was counteracted, then HFD-induced glucose metabolic disorders may show an improvement. Thus, we specifically overexpressed OSMRb in hepatocytes by crossing OSMRbflox/flox mice with mice carrying a Cre recombinase driven by a

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807 808 LoxP LoxP CAT OSMRβ 809 810 Alb-Cre mice Alb-OSMRβ-TG mice 811 CAG promoter OSMRβ LoxP 812 813 B 4 2.85 TG1 TG2 TG3 TG4 NTG Figure 3 OSMRb overexpression in hepato814 2.25 3 1.00 2 1.38 1.47 cytes alleviates hepatic insulin resistance. A: 180kDa 815 OSMRβ 1 Schematic workflow of the establishment of a 816 37kDa GAPDH 0 liver-specific OSMRb-overexpressing TG mouse TG1 TG2 TG3 TG4 NTG 817 strain. B: Immunoblotting of hepatic OSMRb 818 expression in OSMRb-TG mice. GAPDH was used as C 100 D 20 819 10 20 NTG NC a loading control. C: Body weight analysis of NTG TG NC * 820 * 80 8 and OSMRb-TG mice after 24-week NC diet or HFD. NTG HFD 15 15 TG HFD 821 * D: Ameliorated glucose homeostasis in the † 60 6 † * † 10 10 822 † OSMRb-TG mice. Fasting serum glucose and insulin 40 4 823 concentrations were detected at the indicated 5 5 20 2 time points. HOMA-IR was also calculated. E and F: 824 0 0 0 0 Glucose tolerance was assessed by IPGTT (E) and 825 IPITT (F). G: Expression and quantification of the 826 E F indicated proteins in the IRS1/AKT signaling 827 20 25 NTG NC 2 3 pathway. GAPDH was used as a loading control. * * TG NC 828 20 NTG HFD 15 † Data are expressed as means  SD. n Z 10 to 14 * † TG HFD † * 2 * † 829 15 * per group (D); n Z 10 to 14 per group at each † 10 1 *† † 830 * 10 time point (E and F); n Z 3 independent experi* NTG NC 1 † † 5 TG NC 831 Q27 5 ments (G). *P < 0.05 versus the NTG NC mice; NTG HFD y TG HFD 832 0 0 P < 0.05 versus NTG HFD mice. Alb, albumin; 0 0 0 15 30 60 120 0 15 30 60 120 833 AUC, area under the curve; Cre, cyclization Time (min) Time (min) recombination enzyme; GAPDH, glyceraldehyde-3834 phosphate dehydrogenase; HFD, high-fat diet; G 835 NTG HFD TG HFD HOMA-IR, homeostasis model assessment of in836 p-IRS1 180kDa sulin resistance; IPGTT, i.p. glucose tolerance test; 1.5 NTG HFD TG HFD 837 IRS1 180kDa IPITT, i.p. insulin tolerance test; NC, normal chow; † 838 † † NTG, nontransgenic; OSMRb, OSM receptor b p-AKT 60kDa 1 839 chain; TG, transgenic. † AKT 60kDa 840 p-GSK3β 46kDa 0.5 841 GSK3β 46kDa 842 p-FOXO1 72kDa 0 843 FOXO1 72kDa 844 GAPDH 37kDa 845 Insulin Insulin + + + + + + 846 847 848 these results revealed that hepatic OSMRb is required for ½F3 hepatocyte-specific Alb promoter (Figure 3A). Four 849 maintenance of normal glucose and insulin regulation in transgenic mouse lines, TG1 to TG4, were established, and 850 diet-induced obesity. TG4 (hereafter referred to as OSMRb-TG) mice were used 851 for further experiments because TG4 displayed the highest 852 853 OSMRb expression in the liver (Figure 3B). Compared OSMRb Deletion Aggravates Hepatic Steatosis and 854 with non-TG mice, OSMRb-TG mice had significantly Inflammatory Response 855 lower body weights (Figure 3C) and attenuated IR char856 acterized by lower fasting serum glucose and insulin Lipid accumulation in the liver, also known as hepatic 857 concentrations and a lower HOMA-IR index (Figure 3D). steatosis, is strongly associated with hepatic IR,1 which 858 Glucose tolerance and insulin sensitivity were also prompted us to further investigate the impact of genetic 859 improved on OSMRb overexpression (Figure 3, E and F). manipulations to OSMRb on liver morphology and lipid 860 Furthermore, insulin-induced phosphorylation of IRS1, metabolism. After 24 weeks on the HFD, both global and 861 AKT, GSK3b, and FOXO1 was also increased in the liver-restricted OSMRb ablation resulted in heavier livers 862 OSMRb-TG mice, indicating preserved activation of the than in the control mice (Figure 4A and Supplemental ½F4 863 insulin cascade (Figure 3G). Intriguingly, hepatic-specific Figure S3F). Conversely, liver weight remained low on 864 865 overexpression of OSMRb also sufficiently enhanced OSMRb overexpression in hepatocytes compared with that 866 insulin-induced phosphorylation of AKT in adipose tissue of non-TG mice when fed HFD (Figure 4A) and was 867 and skeletal muscle (Supplemental Figure S2B). Thus, associated with improved hepatic steatosis, which was 868

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OSMRb Attenuates Hepatic Steatosis

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assessed by hematoxylin and eosin staining and Oil Red O staining (Figure 4B). On OSMRb deletion (OSMRb-KO) and RNA interference (AdshOSMRb), however, increased lipid droplet accumulation occurred (Figure 4B and Supplemental Figure S3H), which was correlated with a significant increase in hepatic triglycerides, TC, and NEFAs (Figure 4C and Supplemental Figure S3G). In sharp contrast, hepatic triglyceride, TC, and NEFA levels were largely restored in the OSMRb-TG mice (Figure 4C). Consistent with a redirection in the glucose flux toward fatty acid synthesis, we observed that the OSMRb expression level was negatively correlated with mRNA levels of gluconeogenic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in vivo (Figure 4D and Supplemental Figure S3I). Quantitative real-time PCR showed that OSMRb ablation led to decreased levels of genes related to cholesterol efflux (eg, ABCG1) and fatty acid b-oxidation (eg, PPARA and CPT1A) in livers and increased expression levels of genes that regulate

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Figure 4 OSMRb inhibits obesity-induced hepatic steatosis. A: Analysis of LW/BW in the indicated mice strains. B: Representative H&E or Oil Red O staining of liver sections obtained from the indicated mice strains at 24 weeks of HFD feeding. C: Levels of liver triglycerides, TC, and NEFAs were examined in the WT and KO mice, NTG and OSMRb-TG mice on the HFD. D: Quantification of mRNA levels of PEPCK and G6Pc in the experimental conditions described in panels AeC. E and F: mRNA levels of genes related to lipid metabolism were detected via real-time PCR. Data are expressed as means  SD. n Z 8 to 10 per group (A); n Z 8 to 10 per group (C); n Z 6 to 8 per group (E and F). *P < 0.05 versus WT or NTG NC group; yP < 0.05 versus WT or NTG HFD group; z P < 0.05 versus control. G6PC, glucose-6phosphatase; HFD, high-fat diet; H&E, hematoxylin and eosin; KO, knockout; LW/BW, ratio of liver weight to body weight; NC, normal chow; NEFA, nonesterified fatty acid; NTG, nontransgenic; OSMRb, OSM receptor b chain; PEPCK, phosphoenolpyruvate carboxykinase; TC, total cholesterol; TG, transgenic; WT, wild-type.

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cholesterol synthesis (eg, SREBP1c), fatty acid synthesis (eg, ACCA, PPARG, and FAS ), and uptake (eg, CD36 and FABP1) compared with what was found in the livers of the WT or AdshRNA HFD controls (Figure 4E and Supplemental Figure S3J). In addition, OSMRb-KO mice also exhibited aggravated liver damage as evidenced by higher serum concentrations of liver aspartate aminotransferase and alanine aminotransferase (Supplemental Table S3). Accumulation of serum triglycerides, cholesterol, and free fatty acids was also more severe in OSMRb-KO mice (Supplemental Figure S4A). Notably, these adverse effects were reversed on liver-specific OSMRb overexpression (Figure 4F and Supplemental Figure S4B; Supplemental Table S4). Excessive lipid accumulation renders livers more vulnerable to adverse impacts, such as the presence of a proinflammatory mediator in the liver.25 Thus, we assessed whether OSMRb regulates the hepatic inflammatory cytokine milieu in mice fed an HFD with the use of quantitative

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real-time PCR. The OSMRb-KO mice displayed higher levels of proinflammatory cytokines, including IL-1b, IL-6, TNF-a, MCP1, and inducible nitric oxide synthase, and lower levels of anti-inflammatory IL-10 compared with WT controls, whereas OSMRb-TG mice displayed an ameliorative inflammatory response (Supplemental Figure S4, C and D). Concomitantly, the serum concentrations of proinflammatory cytokines (eg, IL-1b, IL-4, IL-6, TNF-a, MCP1) were increased, and anti-inflammatory IL-10 was decreased on OSMRb deletion (Supplemental Table S3), whereas OSMRb-TG mice displayed almost opposite expression concentrations of serum cytokines (Supplemental Table S4). Activation of the NF-kB signaling pathway in the liver can be initiated by obesity and HFD, which leads to downstream cytokine production and subsequent hepatic inflammation.26 We observed that the phosphorylation levels of IKKb, p65, and IkBa, which reflect the activity of NF-kB, were significantly higher in the livers of OSMRbKO mice but lower on OSMRb overexpression compared with that of their respective controls (Figure 5A). The

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OSMRb inhibits obesity-induced inflammation. A: Protein levels in the NF-kB signaling pathway in the livers were assessed using immunoblotting. B: Primary hepatocytes were infected with the indicated adenoviruses before challenging with 0.25 mmol/L palmitate. Immunoblotting and quantification of the NF-kB signaling pathway were performed. Data are expressed as means  SD. n Z 4 per group (A and B). *P < 0.05 versus WT or NTG NC group; y P < 0.05 versus WT or NTG HFD group; zP < 0.05 versus AdshRNA or AdGFP control group; xP < 0.05 versus AdshRNA or AdGFP palmitate group. AdGFP, adenoviral vectors encoding the GFP gene; AdshRNA, adenoviral vectors encoding scrambled shRNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HFD, high-fat diet; KO, knockout; NC, normal chow; NTG, nontransgenic; OSMRb, OSM receptor b chain; TG, transgenic; WT, wild-type.

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negative regulation of the NF-kB signaling pathway by OSMRb was confirmed in primary hepatocytes in vitro (Figure 5B). Taken together, these data clearly demonstrate that OSMRb protects against hepatic steatosis and inflammation in mice.

Hepatic OSMRb Overexpression Improves Metabolism in the Genetic Obesity Model To eliminate the confounding factors of HFD caused by unidentified components, we used a genetic obesity model to determine the biological effect of OSMRb on hepatic metabolism. Leptin-deficient (ob/ob) mice that had developed spontaneous hepatic steatosis24 were injected with adenovirus harboring OSMRb (AdOSMR) through the jugular vein for liver-restricted OSMRb overexpression and fed an NC diet for 4 weeks. Although the body weight remained comparable (Figure 6A), AdOSMRb injections resulted in significantly reduced fasting serum glucose and insulin concentrations and a lower HOMA-IR index compared with that of

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1179 1180 120 20 400 300 1181 AdGFP ob/ob Figure 6 OSMRb overexpression blunts hepatic AdOSMRβ ob/ob * 15 300 1182 insulin resistance, steatosis, and inflammation in 80 * 200 ob/ob mice. A: Body weight analysis of ob/ob mice 1183 * 10 200 injected with AdOSMRb or the AdGFP control. B: 40 1184 100 5 100 OSMRb overexpression in the liver attenuates 1185 0 0 0 0 glucose homeostasis in the ob/ob mice. Fasting 1186 serum glucose and insulin concentrations were C 30 D 20 1187 detected at the indicated time points. HOMA-IR AdGFP ob/ob 3 2 1188 AdOSMRβ ob/ob was also calculated. C and D: The IPGTT (C) and 15 * 1189 20 IPITT (D) assays were performed to evaluate the 2 * 1190 10 * * * 1 insulin sensitivity of mice in the indicated groups. * 10 1191 1 E: Quantification of the body weights of the indi* 5 * AdGFP ob/ob * * * 1192 AdOSMRβ ob/ob cated mice and quantification of the LW/BW in the 0 0 0 0 indicated group. F: Representative H&E or Oil Red Q30 1193 0 15 30 60 120 0 15 30 60 120 Time (min) Time (min) O staining of liver sections obtained from the 1194 AdGFP and AdOSMRb ob/ob groups. G: Levels of 1195 AdGFP ob/ob AdOSMRβ ob/ob E F liver triglycerides, TC, and NEFAs were assessed. H: 1196 6 80 AdGFP ob/ob Quantification of mRNA levels of PEPCK and G6Pc 1197 AdOSMRβ ob/ob 60 using quantitative real-time PCR. I: Quantification 4 1198 * of mRNA levels of proinflammatory and anti40 * 1199 inflammatory markers in the liver. The error bars 2 1200 20 indicate the means  SD. n Z 10 to 14 per group 100 μm 1201 (A); n Z 8 to 12 per group (B and G); n Z 8 to 12 0 0 1202 per group at each time point (C and D); n Z 8 to G 10 per group (I). *P < 0.05 versus AdGFP ob/ob 1203 AdGFP ob/ob 120 30 100 AdOSMRβ ob/ob group. AdGFP, adenoviral vectors encoding the GFP 1204 80 * * 80 20 * gene; AdOSMRb, adenoviruses carrying sequences 1205 60 encoding mouse OSMRb; AUC, area under the 40 1206 40 10 curve; G6PC, glucose-6-phosphatase; HOMA-IR, 20 1207 0 0 0 homeostasis model assessment of insulin resis1208 tance; H&E, hematoxylin and eosin; IPGTT, i.p. H 1.5 I 3 AdGFP ob/ob 1209 glucose tolerance test; IPITT, i.p. insulin tolerance AdGFP ob/ob 1210 * AdOSMRβ ob/ob test; LW/BW, ratio of liver weight to body weight; AdOSMRβ ob/ob 2 1 1211 NEFA, nonesterified fatty acid; PEPCK, phospho* * 1212 enolpyruvate carboxykinase; OSMRb, OSM receptor * * 1 * 0.5 * b chain; TC, total cholesterol. 1213 0 0 1214 IL-6 MCP1 IL-10 PEPCK G6Pc IL-1β TNF-α 1215 1216 1217 adenovirus vectors encoding the GFP gene (AdGFP) controls the cell type and signaling effector.10 Interestingly, both path1218 9,27 (Figure 6B). Glucose regulation and insulin sensitivity were ways are critical regulators of IR and hepatic metabolism. 1219 also improved on OSMRb overexpression (Figure 6, C and To explore how OSMRb alleviates metabolic disorders, we 1220 D). Furthermore, OSMRb overexpression ameliorated the harvested liver tissues from OSMRb-KO and OSMRb-TG 1221 established liver enlargement (Figure 6E) and hepatic lipid mice fed an HFD for 24 weeks. Intriguingly, in vivo genetic 1222 accumulation in ob/ob mice (Figure 6F) and reduced hepatic manipulation did not affect phosphorylation of the MAPK 1223 triglyceride, TC, and NEFA content (Figure 6G). Moreover, family members, which exhibited unaffected activity 1224 gluconeogenesis was inhibited in AdOSMRb mice compared (Supplemental Figure S5A). Nevertheless, the expression 1225 1226 with that of their AdGFP littermates (Figure 6H). We also levels of phosphorylated JAK2 and STAT3, which are crucial 1227 observed significantly lower inflammatory responses in the for the maintenance of glucose homeostasis, were significantly 1228 livers of AdOSMRb-treated mice (Figure 6I). Thus, these reduced on OSMRb deletion and knockdown compared with data indicate that the administration of OSMRb exerts that of the respective controls (Figure 7A and Supplemental ½F7 1229 1230 beneficial effects on IR, hepatic steatosis, and inflammation Figure S5B). Conversely, liver-specific overexpression of 1231 in both diet-induced and genetic obesity. OSMRb in both diet-induced and genetic obesity models 1232 resulted in significant phosphorylation and activation of 1233 JAK2/STAT3 signaling (Figure 7A and Supplemental OSMRb Activates the Hepatic JAK2/STAT3 Signal 1234 Figure S5B). The positive regulation of the JAK2/STAT3 1235 Transduction Pathway cascade by OSMRb was also observed in primary hepatocytes 1236 challenged with palmitate (Figure 7B). These data suggest that 1237 Several signaling pathways, including JAK2/STAT3 and 1238 OSMRb activates the JAK2/STAT3 cascade but not the MAPK, are stimulated by gp130 cytokines, although the 1239 MAPK signaling pathway in the liver of obese mice. spectrum of pathways that are activated varies, depending on 1240

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1303 1241 NC HFD (24 wks) NC HFD (24 wks) A 1304 1242 WT KO WT KO NTG TG NTG TG 1305 1243 1306 1244 p-JAK2 125kDa 1307 1245 JAK2 125kDa 1308 1246 p-STAT3 79kDa 1309 1247 STAT3 79kDa 1310 1248 GAPDH 37kDa 1311 1249 Figure 7 OSMRb activates the JAK2/STAT3 1312 1250 0.9 0.6 pathway in liver. A: Protein levels of total and WT NC TG NC NTG NC KO NC WT HFD TG HFD NTG HFD KO HFD 1313 1251 phosphorylated JAK2 and STAT3 were detected in the mouse livers by immunoblotting. GAPDH was 1314 1252 0.6 0.4 † † * used as a loading control. B: Immunoblotting was 1315 1253 * * † * performed on primary hepatocytes infected with 1316 1254 0.3 0.2 † AdOSMRb or AdshOSMRb and subjected to 0.25 1317 1255 mmol/L palmitate or the control treatment. Data 1318 1256 0 0 are expressed as means  SD. n Z 4 per group. p-JAK2 p-STAT3 p-JAK2 p-STAT3 1319 1257 y *P < 0.05 versus WT NC or NTG NC; P < 0.05 1320 1258 z versus WT HFD or NTG HFD; P < 0.05 versus B Control Palmitate Control Palmitate 1321 1259 AdshRNA or AdGFP control group; xP < 0.05 versus 1322 1260 shRNA shOSMRβ shRNA shOSMRβ AdGFP AdOSMRβ AdGFP AdOSMRβ AdshRNA or AdGFP palmitate group. AdOSMRb, 1323 1261 adenoviruses carrying sequences encoding mouse p-JAK2 125kDa OSMRb; AdshOSMRb, adenoviruses carrying a 1324 1262 JAK2 125kDa shRNA targeting OSMRb; GAPDH, glyceraldehyde1325 1263 3-phosphate dehydrogenase; HFD, high-fat diet; p-STAT3 79kDa 1326 1264 JAK2, Janus kinase 2; KO, knockout; NC, normal 1327 1265 STAT3 79kDa chow; NTG, nontransgenic; OSMRb, OSM receptor b 1328 1266 GAPDH 37kDa chain; TG, transgenic; WT, wild-type. 1329 1267 AdshRNA control AdGFP control 0.3 0.3 AdshOSMRβ control AdOSMRβ control 1330 1268 AdshRNA palmitate AdGFP palmitate AdshOSMRβ palmitate ‡ AdOSMRβ palmitate 1331 1269 § 0.2 0.2 1332 1270 § ‡ § ‡ 1333 1271 ‡ 0.1 0.1 § 1334 1272 1335 1273 0 0 1336 1274 p-JAK2 p-STAT3 p-JAK2 p-STAT3 1337 1275 1338 1276 1339 1277 between the STAT3-TG and OKST mice. Moreover, STAT3 OSMRb Ameliorates Glucose Tolerance, Hepatic 1340 1278 overexpression completely abolished liver enlargement Steatosis, and Inflammation through Activation of 1341 1279 (Figure 8E), lipid accumulation (Figure 8F), hepatic metabolic JAK2/STAT3 Signaling 1342 1280 disorders (Figure 8G and Supplemental Figure S6D), and 1343 1281 Because OSMRb promotes hepatic STAT3 phosphorylation salient inflammation (Supplemental Figure S6E) observed in 1344 1282 in obese mice, we hypothesized that the beneficial role of the OSMRb-KO mice. Thus, STAT3 is a main downstream 1345 1283 OSMRb in glucose homeostasis and hepatic steatosis may be target of OSMRb in obesity-related hepatic steatosis and IR. 1346 1284 attributable to STAT3 activation in the liver. To address this Subsequently, we determined whether OSMRb could 1347 1285 issue, we first examined whether STAT3 exerts similar propreserve its protective biological functions when JAK2/ 1348 1286 1349 1287 tective effects in the absence of OSMRb. Liver-specific STAT3 signaling was blocked. Therefore, we crossed 1350 1288 STAT3 overexpression transgenic mice, designated AlbOSMRb-TG mice with liver-specific STAT3-KO mice to 1351 1289 STAT3-TG mice, were generated with the Cre/LoxP system generate OTSK mice (Supplemental Figure S7A). The 1352 1290 (Supplemental Figure S6A). Alb-STAT3-TG mice were then overexpression of OSMRb and deficiency of STAT3 in the 1353 1291 crossed with OSMRb-KO mice to generate OKST mice liver were confirmed via Western blot analysis (Supplemental 1354 1292 (Supplemental Figure S6B), which were validated via Western Figure S7B). Notably, OSMRb overexpression in the 1355 1293 blot analysis (Supplemental Figure S6C). As expected, absence of STAT3 (OTSK mice) did not protect against 1356 1294 STAT3-TG mice displayed reduced body weight and hepatic steatosis, IR, and hepatic inflammation, which was 1357 1295 improved glucose tolerance compared with control mice observed in OSMRb-TG mice (Figure 9 and Supplemental ½F9 1358 1296 Figure S7, C and D). Instead, OTSK mice displayed a 1359 1297 ½F8 (Figure 8, AeD). More importantly, the deleterious effects of OSMRb ablation were completely abrogated in the presence similar adverse phenotype to that in the STAT3-KO mice fed 1360 1298 1361 1299 of joint STAT3 overexpression as evidenced by the compaan HFD. Together, these data demonstrate that OSMRb 1362 1300 rable body weights (Figure 8A), fasting serum glucose and protects against glucose tolerance, hepatic steatosis, and 1363 1301 insulin (Figure 8B), and insulin tolerance (Figure 8, C and D) inflammation via JAK2/STAT3 signal activation in the liver. 1364 1302

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Figure 8 STAT3 overexpression in the liver mimics the protective effect of OSMRb. A: Analysis of the body weights of the OSMRb-KO, STAT3-TG, and OKST mice fed an HFD. B: Fasting serum glucose and insulin concentrations are detected at the indicated time points, and HOMA-IR was calculated. C and D: Glucose tolerance was assessed by IPGTT (C) and IPITT (D). E: Quantification of the liver weight and ratio of liver weight to body weight in the indicated group. F: Representative H&E or Oil Red O staining of liver sections. G: Levels of liver triglyceride, total cholesterol, and NEFAs were assessed. Data are expressed as means  SD. n Z 9 to 14 per group (A); n Z 8 to 12 per group (B); n Z 8 to 12 per group at each time point (C and D); n Z 9 to 13 per group (E); n Z 8 to 10 per group (G). *P < 0.05 versus control group; yP < 0.05 versus OSMRb-KO group. AUC, area under the curve; HFD, high-fat diet; HOMA-IR, homeostasis model assessment of insulin resistance; H&E, hematoxylin and eosin; IPGTT, i.p. glucose tolerance test; IPITT, i.p. insulin tolerance test; KO, knockout; LW/BW, ratio of liver weight to body weight; NEFA, nonesterified fatty acid; OKST, OSMRb-KO/STAT3-TG; OSMRb, OSM receptor b chain; TG, transgenic.

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Luo et al

Discussion The liver is a key insulin-responsive organ responsible for insulin sensitivity and lipid metabolism. Recent studies have shown that OSMRb is a crucial link in mediating adipose tissue inflammation and IR.19,20 However, the use of global KO mice in these studies increases the difficulty of determining the role of OSMRb in specific tissues in the modulation of local and systemic inflammation and IR. In this study, we depicted the central role of hepatocyte-derived OSMRb in the regulation of insulin sensitivity, hepatic steatosis, and inflammatory response for the first time. Several lines of evidence support this conclusion. First, OSMRb was down-regulated in hepatic tissues in both human samples and mouse models of severe hepatic steatosis. Second, the whole-body deletion of OSMRb aggravated HFD-induced metabolic disorders. Furthermore, liver-restricted OSMRb knockdown with the use of an adenovirus-mediated gene-transfer approach confirmed the deteriorated results. In addition, we established liver-specific OSMRb overexpression TG mice. Maintaining OSMRb expression in the hepatocytes provided a beneficial effect on

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functional glucose tolerance and lipid metabolism. Third, OSMRb phosphorylates and activates the JAK2/STAT3 signaling pathway in the liver. Finally, liver-restricted overexpression of STAT3 rescued glucose tolerance and ameliorated hepatic steatosis and inflammation in the OSMRb-KO mice. Conversely, OSMRb overexpression failed to protect against hepatic steatosis, IR, and hepatic inflammation in the STAT3-deficient mice. We thus propose that hepatocytes are, at least partially, a crucial target for OSMRb-mediated beneficial effects on hepatic glucose homeostasis and lipid metabolism. Therefore, the targeted overexpression of OSMRb in hepatocytes could be a novel therapeutic strategy against obesity-induced metabolic disorders. OSMRb expression is detectable in adipose tissue, primarily in adipose tissue macrophages and liver tissue.19,20 However, the distribution of OSMRb in the liver, especially during the development of metabolic disorders, is less clear. In the current study, we observed that the protein expression of hepatic OSMRb was significantly up-regulated in mice fed an HFD for 8 weeks, but dramatically downregulated in mice challenged with HFD for 16 and 24

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Figure 9 Metabolic protective effect of OSMRb is STAT3 dependent. A: Analysis of the body weights of OSMRb-TG, STAT3-KO, and OTSK mice fed an HFD. B: Fasting serum glucose and insulin concentrations were detected at the indicated time points. HOMA-IR was also calculated. C and D: Glucose tolerance was assessed via IPGTT (C) and IPITT (D). E: Quantification of the liver weights and liver weight to body weight ratio in the indicated group. F: Representative H&E or Oil Red O staining of liver sections. G: Levels of liver triglycerides, total cholesterol, and NEFAs were assessed. Data are expressed as means  SD. n Z 9 to 14 per group (A); n Z 8 to 12 per group (B); n Z 8 to 12 per group at each time point (C and D); n Z 9 to 14 per group (E); n Z 8 to 10 per group (G). *P < 0.05 versus NTG group; yP < 0.05 versus OSMRb-TG group. AUC, area under the curve; HFD, high-fat diet; HOMA-IR, homeostasis model assessment of insulin resistance; H&E, hematoxylin and eosin; IPGTT, i.p. glucose tolerance test; IPITT, i.p. insulin tolerance test; KO, knockout; NEFA, nonesterified fatty acid; NTG, nontransgenic; OSMRb, OSM receptor b chain; OTSK, OSMRb-TG/STAT3-KO; TG, transgenic.

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OSMRb Attenuates Hepatic Steatosis

weeks. Consistently, a previous report showed that OSMRb mRNA expression was elevated in the livers of mice treated with HFD for 8 weeks.19 Furthermore, we demonstrated that HFD induced persistent OSMRb mRNA up-regulation between 8 and 24 weeks. Thus, the decreased protein levels of OSMRb were possibly a result of degradation induced by post-translational modification. Consistent with our observation, OSM expression, which is up-regulated in both adipose and liver tissue in obese mice, has been shown to reduce the protein expression levels of OSMRb in lung fibroblasts by ligand-induced receptor degradation.19,28 Future studies to determine the mechanism of post-transcription regulation of OSMRb expression are required. OSM engages either LIFRa or OSMRb before recruiting gp130 and activating receptor signaling transduction.29 OSM is synthesized in Kupffer cells and macrophages in the liver and has paracrine-like effect on hepatocytes.30 Several of the observed metabolic alterations in OSMRbKO mice are contrary to the known effect of OSM on hepatic metabolism. For example, Henkel et al18 showed that OSM produced by Kupffer cells attenuated insulindependent induction of Akt phosphorylation and

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glucokinase in rat primary hepatocytes and inhibited the expression of key enzymes of hepatic lipid metabolism, such as CPT-1. Notably, human OSM binds to only OSMRb in mice, whereas human OSM recruits both OSMRb and LIFRa in rats and humans.10,31,32 The current study investigated the functional role of OSMRb in OSMRb-KO mice. We observed that OSMRb positively regulates IRS1/Akt phosphorylation in obese mice. The use of OSMRb-KO mice in the current study eliminated the confounding effects of LIFRa in hepatic metabolism. Thus, the distinguishing difference observed between the rat hepatocyte model and OSMRb-KO mice may have been caused by the lack of interaction between OSM and the alternative LIFRa in OSMRb-KO mice. Consistent with our observations, Znoyko et al33 demonstrated that, although OSM was consistently expressed in cirrhotic human liver, OSMRb expression was absent and LIFRa was up-regulated. Therefore, our data suggest that the metabolic function of OSM is attributable to speciesspecific ligand-receptor interactions, and targeting OSMRb may be a more promising therapeutic strategy in humans than targeting OSM.

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Luo et al Furthermore, we showed that liver-specific OSMRboverexpression TG mice exhibited lower fasting serum glucose and insulin concentrations, improved insulin sensitivity, and attenuated hepatic inflammatory responses and dyslipidemia, further indicating that the liver is a crucial insulin-responsive organ responsible for insulin sensitivity and lipid metabolism. Mechanistically, OSMRb expression positively regulates phosphorylation and activation of the downstream JAK2/STAT3 signaling pathway. In support of our data, Inoue et al9 showed that the liver-specific deletion of STAT3 resulted in enhanced IR associated with increased hepatic expression of gluconeogenic genes. We demonstrated that liver-restricted overexpression of STAT3 rescued glucose tolerance and ameliorated hepatic steatosis and inflammation in OSMRb-KO mice, whereas OSMRb overexpression failed to protect against the adverse phenotypes in STAT3-deficient mice. Thus, we further extended these findings by demonstrating that STAT3 is both sufficient and required for OSMRb-mediated metabolic benefits. Moreover, STAT3 is also involved in hepatic ischemia/reperfusion injury,34 regeneration,35 and cancer,36 suggesting a more versatile role of OSMRb in the liver. Intriguingly, we observed that liver-specific overexpression of OSMRb also led to enhanced insulin-induced AKT phosphorylation in adipose tissue and skeletal muscle, suggesting that hepatic OSMRb is required for total body glucose tolerance and IR. Nevertheless, the precise mechanisms underlying the regulation of hepatic OSMRb on total body insulin sensitivity needs further investigation. In addition, although we have shown that liver is a main target of OSMRb, adipose- and skeletal-specific gene manipulations of OSMRb are required in future studies to determine the impact of local OSMRb on IR in these tissues. Our data demonstrate profound salubrious effects of OSMRb in the setting of HFD administration. Of note, a previous study revealed that OSMRb/ mice exhibited IR preceding obesity at 16 weeks of age, suggesting that OSMRb also plays a vital role in maintaining metabolic homeostasis in lean mice.20 However, in the current study, gene manipulations of OSMRb showed no significant effect on IR, hepatic steatosis, and inflammatory response when fed nromal chow. Indeed, overexpression of OSMRb also failed to activate JAK2/STAT3 signaling pathway in lean mice as that observed in ob/ob mice (Figure 6C). We also observed a similar negative result in primary hepatocytes without palmitate challenge. Possible explanations for these discrepancies are the result of the difference in mice, diet composition, or others. Apart from the discrepancy, our results consistently demonstrated the crucial role of OSMRb in maintaining metabolic homeostasis. Taken together, our study suggests that OSMRb expressed by hepatocytes plays a critical role in regulating obesity-induced metabolic disorders, including IR, hepatic steatosis, and inflammatory response. Furthermore, we demonstrated that OSMRb-mediated protection is largely

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STAT3 dependent, at least when comorbid with obesity. In this context, preventing the suppression of the OSMRb/ STAT3 signaling pathway in hepatocytes could be a new strategy for attenuating metabolic disorders.

Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2015.12.028.

References 1. Perry RJ, Samuel VT, Petersen KF, Shulman GI: The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes. Nature 2014, 510:84e91 2. Birkenfeld AL, Shulman GI: Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. Hepatology 2014, 59:713e723 3. Glass CK, Olefsky JM: Inflammation and lipid signaling in the etiology of insulin resistance. Cell Metab 2012, 15:635e645 4. Samuel VT, Shulman GI: Mechanisms for insulin resistance: common threads and missing links. Cell 2012, 148:852e871 5. Berlanga A, Guiu-Jurado E, Porras JA, Auguet T: Molecular pathways in non-alcoholic fatty liver disease. Clin Exp Gastroenterol 2014, 7:221e239 6. Yoon HJ, Cha BS: Pathogenesis and therapeutic approaches for nonalcoholic fatty liver disease. World J Hepatol 2014, 6:800e811 7. Wang XA, Zhang R, Jiang D, Deng W, Zhang S, Deng S, Zhong J, Wang T, Zhu LH, Yang L, Hong S, Guo S, Chen K, Zhang XF, She Z, Chen Y, Yang Q, Zhang XD, Li H: Interferon regulatory factor 9 protects against hepatic insulin resistance and steatosis in male mice. Hepatology 2013, 58:603e616 8. Wang XA, Zhang R, She ZG, Zhang XF, Jiang DS, Wang T, Gao L, Deng W, Zhang SM, Zhu LH, Guo S, Chen K, Zhang XD, Liu DP, Li H: Interferon regulatory factor 3 constrains IKKbeta/NF-kappaB signaling to alleviate hepatic steatosis and insulin resistance. Hepatology 2014, 59:870e885 9. Inoue H, Ogawa W, Ozaki M, Haga S, Matsumoto M, Furukawa K, Hashimoto N, Kido Y, Mori T, Sakaue H, Teshigawara K, Jin S, Iguchi H, Hiramatsu R, LeRoith D, Takeda K, Akira S, Kasuga M: Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo. Nat Med 2004, 10:168e174 10. Richards CD: The enigmatic cytokine oncostatin m and roles in disease. ISRN Inflamm 2013, 2013:512103 11. Richards CD, Brown TJ, Shoyab M, Baumann H, Gauldie J: Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro. J Immunol 1992, 148:1731e1736 12. Chung B, Verdier F, Matak P, Deschemin JC, Mayeux P, Vaulont S: Oncostatin M is a potent inducer of hepcidin, the iron regulatory hormone. FASEB J 2010, 24:2093e2103 13. Miyajima A, Kinoshita T, Tanaka M, Kamiya A, Mukouyama Y, Hara T: Role of Oncostatin M in hematopoiesis and liver development. Cytokine Growth Factor Rev 2000, 11:177e183 14. Yamashita T, Honda M, Nio K, Nakamoto Y, Takamura H, Tani T, Zen Y, Kaneko S: Oncostatin m renders epithelial cell adhesion molecule-positive liver cancer stem cells sensitive to 5-Fluorouracil by inducing hepatocytic differentiation. Cancer Res 2010, 70:4687e4697 15. Han MS, Jung DY, Morel C, Lakhani SA, Kim JK, Flavell RA, Davis RJ: JNK expression by macrophages promotes obesity-induced insulin resistance and inflammation. Science 2013, 339:218e222 16. Priceman SJ, Kujawski M, Shen S, Cherryholmes GA, Lee H, Zhang C, Kruper L, Mortimer J, Jove R, Riggs AD, Yu H: Regulation of adipose tissue T cell subsets by Stat3 is crucial for diet-induced obesity and insulin resistance. Proc Natl Acad Sci U S A 2013, 110: 13079e13084

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OSMRb Attenuates Hepatic Steatosis 17. Grove RI, Mazzucco CE, Radka SF, Shoyab M, Kiener PA: Oncostatin M up-regulates low density lipoprotein receptors in HepG2 cells by a novel mechanism. J Biol Chem 1991, 266: 18194e18199 18. Henkel J, Gartner D, Dorn C, Hellerbrand C, Schanze N, Elz SR, Puschel GP: Oncostatin M produced in Kupffer cells in response to PGE2: possible contributor to hepatic insulin resistance and steatosis. Lab Invest 2011, 91:1107e1117 19. Komori T, Tanaka M, Senba E, Miyajima A, Morikawa Y: Deficiency of oncostatin M receptor beta (OSMRbeta) exacerbates high-fat dietinduced obesity and related metabolic disorders in mice. J Biol Chem 2014, 289:13821e13837 20. Komori T, Tanaka M, Senba E, Miyajima A, Morikawa Y: Lack of oncostatin M receptor beta leads to adipose tissue inflammation and insulin resistance by switching macrophage phenotype. J Biol Chem 2013, 288:21861e21875 21. Li M, Feng B, Wang L, Guo S, Zhang P, Gong J, Zhang Y, Zheng A, Li H: Tollip is a critical mediator of cerebral ischaemia/reperfusion injury. J Pathol 2015, 237:249e262 22. Gomez-Lechon MJ, Donato MT, Martinez-Romero A, Jimenez N, Castell JV, O’Connor JE: A human hepatocellular in vitro model to investigate steatosis. Chem Biol Interact 2007, 165:106e116 23. Zhu LH, Wang A, Luo P, Wang X, Jiang DS, Deng W, Zhang X, Wang T, Liu Y, Gao L, Zhang S, Zhang J, Li H: Mindin/Spondin 2 inhibits hepatic steatosis, insulin resistance, and obesity via interaction with peroxisome proliferator-activated receptor alpha in mice. J Hepatol 2014, 60:1046e1054 24. Hebbard L, George J: Animal models of nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol 2011, 8:35e44 25. Tilg H, Moschen AR: Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 2010, 52:1836e1846 26. Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, Lee J, Shoelson SE: Local and systemic insulin resistance resulting from

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-

27.

28.

29.

30. 31.

32.

33.

34.

35.

36.

hepatic activation of IKK-beta and NF-kappaB. Nat Med 2005, 11: 183e190 Gual P, Le Marchand-Brustel Y, Tanti JF: Positive and negative regulation of insulin signaling through IRS-1 phosphorylation. Biochimie 2005, 87:99e109 Blanchard F, Wang Y, Kinzie E, Duplomb L, Godard A, Baumann H: Oncostatin M regulates the synthesis and turnover of gp130, leukemia inhibitory factor receptor alpha, and oncostatin M receptor beta by distinct mechanisms. J Biol Chem 2001, 276:47038e47045 Caffarel MM, Coleman N: Oncostatin M receptor is a novel therapeutic target in cervical squamous cell carcinoma. J Pathol 2014, 232: 386e390 Elks CM, Stephens JM: Oncostatin m modulation of lipid storage. Biology (Basel) 2015, 4:151e160 Chen SH, Benveniste EN: Oncostatin M: a pleiotropic cytokine in the central nervous system. Cytokine Growth Factor Rev 2004, 15: 379e391 Drechsler J, Grotzinger J, Hermanns HM: Characterization of the rat oncostatin M receptor complex which resembles the human, but differs from the murine cytokine receptor. PLoS One 2012, 7:e43155 Znoyko I, Sohara N, Spicer SS, Trojanowska M, Reuben A: Expression of oncostatin M and its receptors in normal and cirrhotic human liver. J Hepatol 2005, 43:893e900 Matsumoto T, O’Malley K, Efron PA, Burger C, McAuliffe PF, Scumpia PO, Uchida T, Tschoeke SK, Fujita S, Moldawer LL, Hemming AW, Foley DP: Interleukin-6 and STAT3 protect the liver from hepatic ischemia and reperfusion injury during ischemic preconditioning. Surgery 2006, 140:793e802 Debonera F, Aldeguer X, Shen X, Gelman AE, Gao F, Que X, Greenbaum LE, Furth EE, Taub R, Olthoff KM: Activation of interleukin-6/STAT3 and liver regeneration following transplantation. J Surg Res 2001, 96:289e295 He G, Karin M: NF-kappaB and STAT3 - key players in liver inflammation and cancer. Cell Res 2011, 21:159e168

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1861 Supplemental Figure S1 Generation and validation of OSMRb-KO mice. A: Schematic diagram of the targeted exon of mouse OSMRb. Exons are rep1862 resented by black numbered boxes. The target sequence is underlined and highlighted in red. The protospacer adjacent motif sequence is labeled in green. 1863 B: Exemplary results for direct sequencing of founder mice. The black arrowhead marks the start of the mutation. C: Genetic lesions identified in founder mice 1864 derived from individual clones. Genomic DNA from the founder mice was used as template for PCR amplification of the targeted locus. The amplified fragment 1865 Q17 was purified, subcloned into pMD18T vector, and sequenced. D: Representative results of sequencing traces of a WT and a homozygous mouse. The black box 1866 indicates the 17-bp deletion in the homozygous OSMRb-KO mouse; the black line indicates the joint point of the remaining sequence after deleting 17-bp. E: 1867 Q18 A representative Western blot analysis of OSMRb expression in OSMRb-KO mice. GAPDH was used as a loading control. GAPDH, glyceraldehyde-3-phosphate 1868 dehydrogenase; KO, knockout; Mut, mutation; OSMRb, oncostatin M receptor b chain; WT, wild-type. 1869 1870 Supplemental Figure S2 OSMRb positively regulates insulin signaling in adipose tissue and skeletal muscle. WT and OSMRb-KO (A), NTG and OSMRb-TG 1871 (B) mice were fed with HFD for 24 weeks, fasted overnight, and intraperitoneally injected with 0.75 U insulin/kg body weight for 10 minutes. Adipose tissue 1872 and skeletal muscle extracts were immunoblotted with the indicated antibodies. Expression and quantification of the indicated proteins. GAPDH was used as a 1873 loading control. Data are expressed as means  SD. n Z 3 independent experiments. *P < 0.05 versus WT or NTG HFD mice. GAPDH, glyceraldehyde-31874 phosphate dehydrogenase; HFD, high-fat diet; KO, knockout; NTG, nontransgenic; OSMRb, oncostatin M receptor b chain; TG, transgenic; WT, wild-type. 1875 1876 Supplemental Figure S3 Liver OSMRb knockdown aggravates systemic insulin resistance and hepatic steatosis. A: Protein levels of OSMRb in the livers 4 1877 weeks after jugular vein injection of AdshOSMRb. GAPDH was used as a loading control. B: Body weight analysis of mice injected with AdshOSMRb or AdshRNA 1878 controls after HFD feeding. C: Fasting serum glucose and insulin concentrations were detected at the indicated time points. HOMA-IR was also calculated. D 1879 and E: Glucose tolerance was assessed by IPGTT (D) and IPITT (E). F: Analysis of liver weight and its ratio to body weight in the indicated mice strains. 1880 G: Levels of liver triglycerides, TC, and NEFAs were examined in the mice injected with AdshOSMRb or AdshRNA. H: Representative H&E or Oil Red O staining of 1881 liver sections obtained from the indicated mice strains at 24 weeks of HFD feeding. I: Quantification of mRNA levels of PEPCK and G6Pc in the experimental 1882 conditions. J: mRNA levels of genes related to lipid metabolism were detected by real-time PCR. Data are expressed as means  SD. n Z 8 to 12 per group (C); 1883 n Z 10 to 12 per group at each time point (D and E); n Z 8 to 10 per group (F and G); n Z 6 to 8 per group (J). *P < 0.05 versus AdshRNA group (A); 1884 Q19 Q20 yP < 0.05 versus AdshRNA HFD controls (BeJ). Scale bar Z 100 mm. AdshOSMRb, adenovirus carrying a shRNA targeting OSMRb; AdshRNA, adenoviral vectors 1885 encoding scrambled shRNA; AUC, area under the curve; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6PC, glucose-6-phosphatase; HFD, high-fat diet; 1886 HOMA-IR, homeostasis model assessment of insulin resistance; H&E, hematoxylin and eosin; IPGTT, i.p. glucose tolerance test; IPITT, i.p. insulin tolerance 1887 test; KO, knockout; NEFA, nonesterified fatty acid; OSMRb, oncostatin M receptor b chain; PEPCK, phosphoenolpyruvate carboxykinase; TC, total cholesterol; 1888 TG, triglyceride; WT, wild-type. 1889 1890 Supplemental Figure S4 OSMRb inhibits obesity-induced dyslipidemia and inflammation. A and B: Serum concentrations of triglycerides, cholesterol, 1891 and NEFAs were examined in the WT and KO mice (A), NTG and TG mice (B) after HFD feeding for 24 weeks. C and D: Quantification of mRNA levels of 1892 proinflammatory and anti-inflammatory markers in the liver. Data are expressed as means  SD. n Z 8 to 10 per group HFD (A and B); n Z 6 to 9 per group 1893 (C and D). *P < 0.05 versus WT or NTG HFD group. HFD, high-fat diet; KO, knockout; MCP1, monocyte chemoattractant protein-1; NEFA, nonesterified fatty 1894 acid; NTG, nontransgenic; OSMRb, oncostatin M receptor b chain; TG, transgenic; TNF, tumor necrosis factor; WT, wild-type. 1895 1896 Q21 Supplemental Figure S5 OSMRb activates the JAK2/STAT3 pathway but not the MAPK signaling pathway in liver. A: Representative immunoblotting and 1897 quantification of the MAPK signaling pathway in WT, KO, NTG, and TG mice on feeding the NC diet or HFD for 24 weeks. B: Immunoblotting and quantification 1898 Q22 of the hepatic JAK2/STAT3 signaling pathway. Data are expressed as means  SD. n Z 4 per group. *P < 0.05 versus AdshRNA NC or AdGFP lean group; y 1899 P < 0.05 versus AdshRNA NC or AdGFP ob/ob group. 24W, 24 weeks; AdGFP, adenoviral vectors encoding the GFP gene; AdshRNA, adenoviral vectors encoding 1900 scrambled shRNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HFD, high-fat diet; JAK2, Janus kinase 2; KO, knockout; MAPK, mitogen-activated 1901 protein kinase; NC, normal chow; NTG, nontransgenic; OSMRb, oncostatin M receptor b chain; TG, transgenic; WT, wild-type. 1902 1903 Supplemental Figure S6 STAT3 overexpression in the liver mimics the protective effect of OSMRb. Schematic of the generation of STAT3-TG (A) and 1904 OKST mice (B). C: The OSMRb and STAT3 protein levels were detected in the OKST mice by Western blot analysis. D: Quantification of mRNA levels of PEPCK and 1905 G6Pc with quantitative real-time PCR. E: Quantification of mRNA levels of proinflammatory markers in the liver. Data are expressed as means  SD. n Z 8 to 10 1906 per group. *P < 0.05 versus WT HFD group; yP < 0.05 versus OSMRb-KO HFD group. Alb, albumin; Cre, cyclization recombination enzyme; GAPDH, glycer1907 aldehyde-3-phosphate dehydrogenase; G6Pc, glucose-6-phosphatase; HFD, high-fat diet; KO, knockout; MCP1, monocyte chemoattractant protein-1; OKST, 1908 OSMRb-KO/STAT3-TG; OSMRb, oncostatin M receptor b chain; PEPCK, phosphoenolpyruvate carboxykinase; TG, transgenic; TNF, tumor necrosis factor; WT, wild1909 type. 1910 1911 Supplemental Figure S7 Metabolic protective effect of OSMRb is STAT3-dependent. A: Schematic of the generation of Alb-STAT3-KO and OTSK mice. 1912 B: OSMRb and STAT3 protein levels were detected in the OTSK mice via Western blot analysis. C: Quantification of mRNA levels of PEPCK and G6Pc using 1913 quantitative real-time PCR. D: Quantification of mRNA levels of proinflammatory markers in the liver. Data are expressed as means  SD. n Z 8 to 10 per 1914 group. *P < 0.05 versus NTG HFD group; yP < 0.05 versus OSMRb-TG HFD group. Alb, albumin; Cre, cyclization recombination enzyme; GAPDH, glyceraldehyde1915 3-phosphate dehydrogenase; G6Pc, glucose-6-phosphatase; HFD, high-fat diet; KO, knockout; MCP1, monocyte chemoattractant protein-1; NTG, non1916 transgenic; OSMRb, oncostatin M receptor b chain; OTSK, OSMRb-TG/STAT3-KO; PEPCK, phosphoenolpyruvate carboxykinase; TG, transgenic; TNF, tumor ne1917 crosis factor; WT, wild-type.

FLA 5.4.0 DTD  AJPA2293_proof  19 March 2016  1:16 am  EO: AJP15_0501

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