Resistin does not down-regulate the transcription of insulin receptor promoter

Resistin does not down-regulate the transcription of insulin receptor promoter 1 first-author †E-mail:qiao.xiaozhi@163.comgbbf2000@sina.com †E-mail:qiao.xiaozhi@163.comgbbf2000@sina.com qiao.xiaozhi@163.com 2 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:qiao.xiaozhi@163.comgbbf2000@sina.com †E-mail:qiao.xiaozhi@163.comgbbf2000@sina.com qiao.xiaozhi@163.com Department of Very Important Person, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Very Important Person, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Objective: 2 To detect the effect of resistin on the transcription of insulin receptor promoter. Methods: 2 Luciferase reporter gene was fused downstream of human insulin receptor promoter and the enzymatic activity of luciferase was determined in the presence or absence of resistin. The resistin expressed with plasmid was stained with antibody against Myc tag which was in frame fused with resistin coding sequence, and then imaged with confocal microscopy. Results: 2 The treatment of pIRP-LUC transfected cells with recombinant resistin did not result in significant difference in the enzymatic activity of luciferase compared to the untreated cells. Cell staining showed that green fluorescence could be observed in the cytoplasm, but not in the nucleus. Conclusion: 2 The results suggest that the endogenous resistin may functionally locate in the cytoplasm, but does not enter the nucleus and not down-regulate the transcription of insulin receptor promoter. Resistin Insulin receptor promoter Luciferase reporter gene INTRODUCTION 1 Type 2 diabetes (T2D) is a condition that is expected to affect over 300 million people worldwide by 2025 (Adeghate et al., 2006 2006 ). Although T2D has a multi-factorial etiology, one of the well-established risk factors for T2D is obesity (Proietto, 2005 2005 ). Recently, adipose tissue was considered to not only act as an energy reservoir, but also emerge as a key secretary organ (Kershaw and Flier, 2004 2004 ), releasing a number of bioactive molecules such as leptin, adiponectin, tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor type 1, and the recently described hormone resistin (Steppan et al., 2001a 2001a ). Resistin, a cysteine-rich hormone belonging to the newly discovered resistin-like molecule (RELM) or found in the inflammatory zone (FIZZ) family (Holcomb et al., 2000 2000 ; Kim et al., 2001 2001 ), has been proposed to form a biochemical link between obesity and T2D (Steppan et al., 2001a 2001a ; 2001b 2001b ). In vivo and in vitro studies have demonstrated that high concentrations of resistin impair insulin sensitivity and cause insulin resistance. In the diet-induced obese mice, immunoneutralization of resistin led to a 20% drop in blood glucose, and improved insulin sensitivity (Muse et al., 2004 2004 ). In contrast, recombinant resistin impairs the insulin-stimulated glucose uptake (Banerjee et al., 2004 2004 ). Moreover, overexpression of resistin led to inhibition of adipocyte differentiation (Rajala et al., 2003 2003 ) and impaired glucose tolerance in hepatocytes (Zhou et al., 2006 2006 ), implying that resistin induces insulin resistance. However, the mechanism of resistin in inducing insulin resistance remains poorly understood. It was reported that resistin down-regulated mRNA and protein expression of insulin receptor in rodent pancreatic beta-cells, but did not affect insulin secretion (Brown et al., 2007 2007 ). These results indicate that resistin could lead to insulin resistance in mRNA and protein levels, but the resistin effect on insulin receptor in DNA level cannot be concluded. To detect whether insulin receptor promoter could be activated by exogenous resistin, luciferase reporter gene was fused downstream of human insulin receptor promoter and the enzymatic activity of luciferase was determined in the presence or absence of resistin. MATERIALS AND METHODS 1 Reagents 2 Plasmid pSecTag2B, lipofectamine 2000, Opti-MEM, Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were bought from Invitrogen (Gibco, USA). Monoclonal rabbit anti-Myc antibody and fluorescein isothiocyanate (FITC) [or horseradish peroxidase (HRP)]-labeled goat anti-rabbit IgG (H+L) were from Beyotime® Institute of Biotechnology, China. All the restriction enzymes, VentR® DNA polymerase, DNA marker 2000 and T4 DNA ligase were purchased from New England Biolabs, UK. Primers were synthesized by Invitrogen. Luciferase assay system and pGL3-control were from Promega, USA. phINSRP-1, a plasmid containing a 1.8 kb promoter of human insulin receptor, was the generous gift from Dr. Graeme Bell (University of Chicago, IL, USA). Recombinant resistin protein was from Pepro Tech Asia (USA). Protein marker and enhanced chemiluminescence (ECL) were from Hou-Bio Tech. Co., Ltd., Hong Kong, China. Cell culture 2 The normal human hepatic cells (L-02) were maintained by DMEM containing 10% FBS, and placed in the incubator under 5% CO 2 at 37 °C. The medium was freshened once every two days and the cells were passaged every three days. Constructs 2 pSecTag2B-RETN was described previously (Qiao et al., 2007a 2007a ). To develop pCMV-LUC, luciferase coding sequence was PCR amplified with forward (5′-GAT CGG ATC CGC CAC CAT GGA A-3′) and reverse (5′-GAC TCT CGA GCC ACG GCG ATC TTT-3′) primers from pGL3-control plasmid and digested with Bam HI/ Xho I, followed by insertion into pSecTag2B vector. pCMV-LUC was restricted with Spe I/ Nhe I to remove CMV promoter, and the construct was self-ligated to produce plasmid p0-LUC, in which the expression of reporter luciferase was not driven by any promoter. The promoter of insulin receptor (IRP) was amplified by PCR with forward (5′-GGG GGC TAG CGG CCA TTG CAC TCC A-3′) and reverse (5′-AAT TGG ATC CTG CGG GAG CGC GGG G-3′) primers taking phINSRP-1 as the template. The PCR products were digested with Nhe I/ Bam HI and inserted into pCMV-LUC that was restricted with Spe I/ Bam HI, yielding pIRP-LUC. All the constructs were confirmed by sequencing. Transfection 2 L-02 cells were trypsinized and seeded with fresh medium without antibiotics at a density of 1.0×10 5 per well in a 24-well plate. After 24 h, cells were transfected with p0-LUC, pCMV-LUC and pIRP-LUC, respectively, according to the protocol provided by the manufacturer with some modifications. Three microlitres of lipofectamine 2000 was mixed with 47 µl of Opti-MEM and incubated for 5 min at room temperature. In another tube, 1 µg of luciferase-expressing plasmid was mixed with 50 µl of Opti-MEM. These two solutions were combined, mixed by tapping, and incubated for 20 min at room temperature. Cells were washed with prewarmed phosphate buffered saline (PBS), and then 100 µl Opti-MEM was added. The solution containing lipofectamine 2000 and plasmids was then added to the wells. After incubation for 4 h at 37 °C, growth medium was replaced with DMEM containing 10% FBS and cells were incubated further. Immunofluorescence 2 L-02 cells were seeded in the 24-well plate containing a round cover slip overnight at 37 °C in serum-containing medium until they reached 50% confluence. After transfection with pSecTag2B-RETN, the cells were incubated for further 24 h. The cells were washed twice with PBS and fixed with 100% methanol for 5 min at room temperature, followed by blocking with PBS containing 10% defatted dry milk (blocking solution) for 20 min at room temperature. Anti-Myc (C-term)-FITC antibody, diluted in the blocking solution (1:500), was added and incubated for 1 h in the dark at room temperature, and then imaged with confocal microscopy. Western blot analysis 2 Whole cell lysate extracted from transfected L-02 cells (6-well) was separated by 15% SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride (PVDF) membranes (Schleicher & Schuell, German), and immunodetected using rabbit anti-Myc antibody and goat anti-rabbit IgG-HRP conjugate. Bands were visualized with an ECL Western blot analysis system (Beyotime® Institute of Biotechnology, China). Luciferase detection 2 L-02 cells were grown to 70% or 80% confluence on 24-well plates. Cells were transiently transfected by lipofectamine 2000 with 0.8 µg of pIRP-LUC, pCMV-LUC or p0-LUC, respectively. Recombinant resistin protein was added into wells 4 h post-transfection. Cell extracts were prepared 48 h after transfection, and the luciferase activity assay was performed with the reporter assay kits (Beyotime®Institute of Biotechnology, China). To correct for variability in transfection efficiency, each experiment was performed for at least 3 times, and each transfection was carried out in triplicate. Statistical analysis 2 Group data are expressed as mean± SEM . Data were compared between experimental groups by two-way ANOVA. Differences between groups were further evaluated by Fisher-protected least squares differences. Differences were considered significant at a value of P < 0.05. RESULTS 1 Plasmid preparation 2 To detect the effect of resistin on the transcription of IRP, three luciferase-expressing vectors were constructed. In plasmid pIRP-LUC, the expression of luciferase reporter was controlled with IRP. When resistin was added to cells transfected with pIRP-LUC, resistin effect on IRP could be deduced by the enzyme activity of luciferase. pCMV-LUC will produce luciferase under CMV promoter, and there is no promoter to drive luciferase expression in p0-LUC. pCMV-LUC produced a band corresponding to 1700 bp after digestion with Bam HI/ Xho I, suggesting that luciferase coding sequence was inserted into pSecTag2B correctly. The bands about 1800 bp and 800 bp were released from pIRP-LUC and pCMV-LUC when restricted with Bam HI/ Bgl II, respectively, indicating that CMV promoter was replaced with IRP. No band was found after the treatment of plasmid p0-LUC with Bam HI/ Bgl II. All the plasmids were confirmed by sequencing and prepared with QIAGEN plasmid Miniprep Kit for transfection. Effect of recombinant resistin on IRP transcription 2 In the above constructs luciferase was fused in frame with the Myc tag of pSecTag2B vector. To detect luciferase expression, total cell lysates were prepared and blotted with monoclonal rabbit anti-Myc antibody and subsequent HRP-labeled goat anti-rabbit IgG (H+L). Immune complexes were detected by enhanced chemiluminescence. A target band of about 61 kD was found from pIRP-LUC and pCMV-LUC-transfected cells when blotted with anti-Myc antibody (Fig. 1 1 ), suggesting that luciferase was successfully expressed under the control of CMV or IRP promoter. The lysate from p0-LUC transfected cells did not produce bands. Cell extracts were prepared from pIRP-LUC, pCMV-LUC and p0-LUC transfected cells, respectively, and subjected for Western blot with anti-Myc antibody. A band of about 61 kD was found in transfected cells with either pIRP-LUC (Lane 1) or pCMV-LUC (Lane 2), suggesting that luciferase was successfully expressed under the control of CMV or IRP promoter. Luciferase was not expressed in the cells transfected with p0-LUC which has no promoter (Lane 3) To detect the enzymatic activity of luciferase, luciferase-expressing plasmids were transfected and 4 h later recombinant resistin was added. Cell lysate was prepared 48 h post-transfection. Luciferase activity in cell lysates was determined with a luciferase assay kit and normalized to total cell protein as measured by bicinchoninic acid (BCA) assay kit. When treated with resistin protein, the enzymatic activity of pIRP-LUC transfected cells did not show significant difference compared to the untreated cells, suggesting that the transcription of IRP could not be activated by the added resistin (Fig. 2 2 ). Unexpectedly, luciferase activity of pCMV-LUC transfected cells increased significantly in the presence of resistin when compared to that in the absence of resistin, the reason of which remains to be investigated. The cells transfected with p0-LUC showed much lower enzymatic activity, either in the presence or absence of resistin. Luciferase-expressing plasmids driven by different promoters were transfected into L-02 cells, followed by the treatment of recombinant resistin or not. Cells were lysed and enzymatic activity of luciferase was detected 48 h post-transfection. Both pIRP-LUC and p0-LUC transfected cells did not show significant luciferase activity in the presence of resistin compared to that in the untreated cells ( P > 0.05). The treatment of pCMV-LUC with resistin significantly increased the luciferase activity ( P < 0.05). RLU: Relative light unit Expressed resistin in the cytoplasm 2 Due to the finding that IRP transcription could not be activated by recombinant resistin, the endogenous resistin expressed with plasmid was stained with antibody against Myc tag that was in frame fused with the resistin coding sequence. To this end, resistin-expressing plasmid pSecTag2B-RETN was developed as described previously and transfected into L-02 cells. To detect the expression of resistin, cell lysate was prepared as above and the resistin protein was blotted with anti-Myc antibody in Western blot. The finding of a band corresponding to 13 kD indicates that resistin protein was expressed correctly (Fig. 3 3 ). For immunostaining, cells were incubated with monoclonal rabbit anti-Myc antibody and FITC-labeled goat anti-rabbit IgG (H+L) in sequence 48 h post-transfection. Green fluorescence could be observed in the cytoplasm, but not in the nucleus (Fig. 4 4 ). The results suggest that endogenous resistin may functionally locate in the cytoplasm but not enter the cell nucleus, corresponding to the above finding that IRP promoter could not be activated with recombinant resistin. L-02 cells were transfected with resistin-expressing plasmid and the cell lysate was extracted for detecting the expression of resistin with Western blot of anti-Myc antibody. Lanes 1 and 2: pSecTag2B-RETN transfected L-02 cells; Lane 3: Un-transfected L-02 cells pSecTag2B-RETN was transfected into L-02 cells and the expression was detected with immunostaining. (a), (b), (c) Green fluorescence was observed in the cytoplasm, but not in the nucleus (20× magnification), suggesting that expressed resistin locates in L-02 cytoplasm; (d), (e), (f) Green fluorescence was not detected in un-transfected L-02 cells (20× magnification). (a), (d) Merge channel; (b), (e) 488 nm channel; (c), (f) Transmission channel DISCUSSION 1 Resistin belongs to the family of resistin-like molecules, and the resistin mRNA encodes a 114-amino acid polypeptide that contains a 20-amino acid signal sequence (Ukkola, 2002 2002 ). Recent evidence has demonstrated that resistin is not expressed exclusively in adipose tissue, with at least one group demonstrating its expression in islet tissue and clonal β-cells (Minn et al., 2003 2003 ). Resistin expression has also been identified in the liver and it has been suggested that resistin is rather in liver disease and may induce insulin resistance in humans (Yagmur et al., 2006 2006 ). In previous studies, we found elevated resistin level in serum with diabetes and coronary artery diseases (Qiao et al., 2007b 2007b ), which was accordant with several other researches. For example, Tokuyama et al. ( 2007 2007 ) reported that serum resistin concentrations were significantly higher in the insulin-resistant subgroup compared with the insulin-sensitive subgroup and correlated with insulin sensitivity; Al-Sari et al. ( 2007 2007 ) found that resistin levels are higher in diabetic patients and positively correlated with body mass. In lean subjects, the resistin levels of serum have been reported to be approximately 5~15 ng/ml (Al-Daghri et al., 2005 2005 ; Gerber et al., 2005 2005 ), whereas in either T2D or obesity, the resistin levels increase to as high as 40 ng/ml (Fehmann and Heyn, 2002 2002 ). These findings show that resistin might represent the long-sought link between obesity and insulin resistance. Studies reported that resistin inhibits glucose uptake and impairs glucose tolerance, inducing insulin resistance via affecting insulin signal transduction pathway (Fan et al., 2007 2007 ; Liu et al., 2008 2008 ; Zhou et al., 2006 2006 ). However, the relationship between insulin and insulin receptor was still poorly understood. The interaction of resistin and insulin receptor may be resulted from three factors as follows: (1) In DNA level, resistin down-regulates the promoter activity of insulin receptor; (2) In RNA level, resistin decreases mRNA expression of insulin receptor; (3) In protein level, resistin affects the activity of insulin receptor protein through direct or indirect interaction with insulin receptor protein. In the present study, the treatment of pIRP-LUC transfected cells with 100 ng/ml resistin protein did not result in significant difference in enzymatic activity compared to the untreated cells, suggesting that the transcription of IRP could not be activated by the added resistin. However, Brown et al. ( 2007 2007 ) reported that incubation with 40 ng/ml resistin significantly decreased insulin receptor mRNA and protein expression, but did not affect insulin secretion in mice. Liu et al.( 2006 2006 ) found that rat resistin concentrates in the cytoplasm around cell nucleus. To determine the location of human resistin in hepatocytes, we transfected L-02 cells with human resistin-expressing plasmid and found that the expressed resistin located in the cytoplasm in cell-staining, but not in the nucleus, confirming that human resistin does not down-regulate the transcription of insulin receptor in DNA level. It is not known whether rat resistin decreased the promoter activity of insulin receptor in mice. Species differences between mice and humans, or the cell specificity of hepatocytes and rodent pancreatic beta-cells may be other factors in the insulin resistance-based resistin. Further studies should be performed. Interestingly, luciferase activity of pCMV-LUC transfected cells increased significantly in the presence of resistin when compared to that in the absence of resistin protein, the reason of which remains to be investigated. CONCLUSION 1 In summary, we determined the promoter activity of insulin receptor in the presence or absence of recombinant resistin by detecting the enzymatic activity of luciferase. The results indicate that resistin does not down-regulate the receptor promoter. Further studies of resistin function on insulin resistance will facilitate the development of therapeutic blockers in diabetic patients, and provide a new effective therapy for metabolic syndrome, T2D, and a series of cardiovascular diseases related to diabetes. References E Adeghate , P Schattner , E Dunn . An update on the etiology and epidemiology of diabetes mellitus . Ann N Y Acad Sci , 2006 . 1084 ( 1 ): 1 - 29 . 17151290 DOI: 10.1196/annals.1372.029 http://doi.org/10.1196/annals.1372.029 . An update on the etiology and epidemiology of diabetes mellitus N Al-Daghri , R Chetty , PG McTernan , K Al-Rubean , O Al-Attas , AF Jones , S Kumar . Serum resistin is associated with C-reactive protein and LDL cholesterol in type 2 diabetes and coronary artery disease in a Saudi population . Cardiovasc Diabetol , 2005 . 4 ( 1 ): 10 15998471 DOI: 10.1186/1475-2840-4-10 http://doi.org/10.1186/1475-2840-4-10 . Serum resistin is associated with C-reactive protein and LDL cholesterol in type 2 diabetes and coronary artery disease in a Saudi population IE Al-Sari , FA Al-Quobaili , YM Kabalan . Serum resistin levels in Syrian obese patients with diabetes mellitus type II . Saudi Med J , 2007 . 28 ( 12 ): 1890 - 1894 . 18060223 . Serum resistin levels in Syrian obese patients with diabetes mellitus type II RR Banerjee , SM Rangwala , JS Shapiro , AS Rich , B Rhoades , Y Qi , J Wang , MW Rajala , A Pocai , PE Scherer , 等 . Regulation of fasted blood glucose by resistin . Science , 2004 . 303 ( 5661 ): 1195 - 1198 . 14976316 DOI: 10.1126/science.1092341 http://doi.org/10.1126/science.1092341 . Regulation of fasted blood glucose by resistin JE Brown , DJ Onyango , SJ Dunmore . Resistin down-regulates insulin receptor expression, and modulates cell viability in rodent pancreatic beta-cells . FEBS Lett , 2007 . 581 ( 17 ): 3273 - 3276 . 17597619 DOI: 10.1016/j.febslet.2007.06.031 http://doi.org/10.1016/j.febslet.2007.06.031 . Resistin down-regulates insulin receptor expression, and modulates cell viability in rodent pancreatic beta-cells HQ Fan , N Gu , F Liu , L Fei , XQ Pan , M Guo , RH Chen , XR Guo . Prolonged exposure to resistin inhibits glucose uptake in rat skeletal muscles . Acta Pharmacol Sin , 2007 . 28 ( 3 ): 410 - 416 . 17303005 DOI: 10.1111/j.1745-7254.2007.00523.x http://doi.org/10.1111/j.1745-7254.2007.00523.x . Prolonged exposure to resistin inhibits glucose uptake in rat skeletal muscles HC Fehmann , J Heyn . Plasma resistin levels in patients with type 1 and type 2 diabetes mellitus and in healthy controls . Horm Metab Res , 2002 . 34 ( 11-12 ): 671 - 673 . 12660880 DOI: 10.1055/s-2002-38241 http://doi.org/10.1055/s-2002-38241 . Plasma resistin levels in patients with type 1 and type 2 diabetes mellitus and in healthy controls M Gerber , A Boettner , B Seidel , A Lammert , J Bar , E Schuster , J Thiery , W Kiess , J Kratzsch . Serum resistin levels of obese and lean children and adolescents: biochemical analysis and clinical relevance . J Clin Endocrinol Metab , 2005 . 90 ( 8 ): 4503 - 4509 . 15928242 DOI: 10.1210/jc.2005-0437 http://doi.org/10.1210/jc.2005-0437 . Serum resistin levels of obese and lean children and adolescents: biochemical analysis and clinical relevance IN Holcomb , RC Kabakoff , B Chan , TW Baker , A Gurney , W Henzel , C Nelson , HB Lowman , BD Wright , NJ Skelton , 等 . FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family . EMBO J , 2000 . 19 ( 15 ): 4046 - 4055 . 10921885 DOI: 10.1093/emboj/19.15.4046 http://doi.org/10.1093/emboj/19.15.4046 . FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family EE Kershaw , JS Flier . Adipose tissue as an endocrine organ . J Clin Endocrinol Metab , 2004 . 89 ( 6 ): 2548 - 2556 . 15181022 DOI: 10.1210/jc.2004-0395 http://doi.org/10.1210/jc.2004-0395 . Adipose tissue as an endocrine organ KH Kim , K Lee , YS Moon , HS Sul . A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation . J Biol Chem , 2001 . 276 ( 14 ): 11252 - 11256 . 11278254 DOI: 10.1074/jbc.C100028200 http://doi.org/10.1074/jbc.C100028200 . A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation F Liu , XR Guo , HX Gong , YH Ni , L Fei , XQ Pan , M Guo , RH Chen . A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation . Chin Med J (Engl) , 2006 . 119 ( 6 ): 496 - 503 . 16584648 . A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation F Liu , T Yang , B Wang , M Zhang , N Gu , J Qiu . Resistin induces insulin resistance, but does not affect glucose output in rat-derived hepatocytes . Acta Pharmacol Sin , 2008 . 29 ( 1 ): 98 - 104 . 18158871 . Resistin induces insulin resistance, but does not affect glucose output in rat-derived hepatocytes AH Minn , NB Patterson , S Pack , SC Hoffmann , O Gavrilova , C Vinson , DM Harlan , A Shalev . Resistin is expressed in pancreatic islets . Biochem Biophys Res Commun , 2003 . 310 ( 2 ): 641 - 645 . 14521959 DOI: 10.1016/j.bbrc.2003.09.061 http://doi.org/10.1016/j.bbrc.2003.09.061 . Resistin is expressed in pancreatic islets ED Muse , S Obici , S Bhanot , BP Monia , RA McKay , MW Rajala , PE Scherer , L Rossetti . Role of resistin in diet-induced hepatic insulin resistance . J Clin Invest , 2004 . 114 ( 2 ): 232 - 239 . 15254590 DOI: 10.1172/JCI200421270 http://doi.org/10.1172/JCI200421270 . Role of resistin in diet-induced hepatic insulin resistance J Proietto . Mechanisms of insulin resistance caused by nutrient toxicity . Hepatol Res , 2005 . 33 ( 2 ): 87 - 91 . 16203173 DOI: 10.1016/j.hepres.2005.09.010 http://doi.org/10.1016/j.hepres.2005.09.010 . Mechanisms of insulin resistance caused by nutrient toxicity XZ Qiao , XF Wang , DJ Zhao , ZR Xu , LD Yu , Z Chen , YM Yang . Assembly of full-length human resistin gene in vitro and construction of its eukaryotic expression vector . J Zhejiang Univ (Med Ed) , 2007 . 36 ( 6 ): 588 - 591 . (in Chinese) . Assembly of full-length human resistin gene in vitro and construction of its eukaryotic expression vector XZ Qiao , YM Yang , ZR Xu , 等 . Relationship between resistin level in serum and acute coronary syndrome or stable angina pectoris . J Zhejiang Univ Sci B , 2007 . 8 ( 12 ): 875 - 880 . DOI: 10.1631/jzus.2007.B0875 http://doi.org/10.1631/jzus.2007.B0875 . Relationship between resistin level in serum and acute coronary syndrome or stable angina pectoris MW Rajala , S Obici , PE Scherer , L Rossetti . Adipose-derived resistin and gut-derived resistin-like molecule—beta selectively impair insulin action on glucose production . J Clin Invest , 2003 . 111 ( 2 ): 225 - 230 . 12531878 DOI: 10.1172/JCI16521 http://doi.org/10.1172/JCI16521 . Adipose-derived resistin and gut-derived resistin-like molecule—beta selectively impair insulin action on glucose production CM Steppan , ST Bailey , S Bhat , EJ Brown , RR Banerjee , CM Wright , HR Patel , RS Ahima , MA Lazar . The hormone resistin links obesity to diabetes . Nature , 2001 . 409 ( 6818 ): 307 - 312 . 11201732 DOI: 10.1038/35053000 http://doi.org/10.1038/35053000 . The hormone resistin links obesity to diabetes CM Steppan , EJ Brown , CM Wright , S Bhat , RR Banerjee , CY Dai , GH Enders , DG Silberg , X Wen , GD Wu , 等 . A family of tissue-specific resistin-like molecules . Proc Natl Acad Sci USA , 2001 . 98 ( 2 ): 502 - 506 . DOI: 10.1073/pnas.98.2.502 http://doi.org/10.1073/pnas.98.2.502 . A family of tissue-specific resistin-like molecules Y Tokuyama , H Osawa , T Ishizuka , H Onuma , K Matsui , T Egashira , H Makino , A Kanatsuka . Serum resistin level is associated with insulin sensitivity in Japanese patients with type 2 diabetes mellitus . Metabolism , 2007 . 56 ( 5 ): 693 - 698 . 17445546 DOI: 10.1016/j.metabol.2006.12.019 http://doi.org/10.1016/j.metabol.2006.12.019 . Serum resistin level is associated with insulin sensitivity in Japanese patients with type 2 diabetes mellitus O Ukkola . Resistin—a mediator of obesity-associated insulin resistance or an innocent bystander? . Eur J Endocrinol , 2002 . 147 ( 5 ): 571 - 574 . 12444887 DOI: 10.1530/eje.0.1470571 http://doi.org/10.1530/eje.0.1470571 . Resistin—a mediator of obesity-associated insulin resistance or an innocent bystander? E Yagmur , C Trautwein , AM Gressner , F Tacke . Resistin serum levels are associated with insulin resistance, disease severity, clinical complications, and prognosis in patients with chronic liver diseases . Am J Gastroenterol , 2006 . 101 ( 6 ): 1244 - 1252 . 16771945 DOI: 10.1111/j.1572-0241.2006.00543.x http://doi.org/10.1111/j.1572-0241.2006.00543.x . Resistin serum levels are associated with insulin resistance, disease severity, clinical complications, and prognosis in patients with chronic liver diseases L Zhou , Y Li , T Xia , S Feng , X Chen , Z Yang . Resistin overexpression impaired glucose tolerance in hepatocytes . Eur Cytokine Netw , 2006 . 17 ( 3 ): 189 - 195 . 17194639 . Resistin overexpression impaired glucose tolerance in hepatocytes We would like to thank Dr. Graeme Bell, University of Chicago, USA, for the generous gift of plasmid phINSRP-1. 10.1631/jzus.B0710637 Project supported by the National 11th Five-Year Plan of Scientific and Technological Program (No. 2006BAI02B08) of China, and the Department of Science and Technology of Zhejiang Province (No. 2003C33031), China 2008-01-13 2008-01-21 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 09