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• W2072676249 abstract "Increased serum levels of resistin, a molecule secreted by fat cells, have been proposed as a possible mechanistic link between obesity and insulin resistance. To further investigate the effects of resistin on glucose metabolism, we derived a novel transgenic strain of spontaneously hypertensive rats expressing the mouse resistin gene under the control of the fat-specific aP2 promoter and also performed in vitro studies of the effects of recombinant resistin on glucose metabolism in isolated skeletal muscle. Expression of the resistin transgene was detected by Northern blot analysis in adipose tissue and by real-time PCR in skeletal muscle and was associated with increased serum fatty acids and muscle triglycerides, impaired skeletal muscle glucose metabolism, and glucose intolerance in the absence of any changes in serum resistin concentrations. In skeletal muscle isolated from non-transgenic spontaneously hypertensive rats, in vitro incubation with recombinant resistin significantly inhibited insulin-stimulated glycogenesis and reduced glucose oxidation. These findings raise the possibility that autocrine effects of resistin in adipocytes, leading to release of other prodiabetic effector molecules from fat and/or paracrine actions of resistin secreted by adipocytes embedded within skeletal muscle, may contribute to the pathogenesis of disordered skeletal muscle glucose metabolism and impaired glucose tolerance. Increased serum levels of resistin, a molecule secreted by fat cells, have been proposed as a possible mechanistic link between obesity and insulin resistance. To further investigate the effects of resistin on glucose metabolism, we derived a novel transgenic strain of spontaneously hypertensive rats expressing the mouse resistin gene under the control of the fat-specific aP2 promoter and also performed in vitro studies of the effects of recombinant resistin on glucose metabolism in isolated skeletal muscle. Expression of the resistin transgene was detected by Northern blot analysis in adipose tissue and by real-time PCR in skeletal muscle and was associated with increased serum fatty acids and muscle triglycerides, impaired skeletal muscle glucose metabolism, and glucose intolerance in the absence of any changes in serum resistin concentrations. In skeletal muscle isolated from non-transgenic spontaneously hypertensive rats, in vitro incubation with recombinant resistin significantly inhibited insulin-stimulated glycogenesis and reduced glucose oxidation. These findings raise the possibility that autocrine effects of resistin in adipocytes, leading to release of other prodiabetic effector molecules from fat and/or paracrine actions of resistin secreted by adipocytes embedded within skeletal muscle, may contribute to the pathogenesis of disordered skeletal muscle glucose metabolism and impaired glucose tolerance. In industrialized societies, type 2 diabetes is a common cause of morbidity and mortality that is characterized by insulin resistance often in association with central obesity. However, the mechanisms that underlie the widely recognized relationship between obesity and insulin resistance remain to be defined. Although skeletal muscles are quantitatively the most important site of insulin-stimulated glucose disposal (1DeFronzo D.J. Bonadonna R.C. Ferrannini E. Diabetes Care. 1992; 15: 318-368Crossref PubMed Scopus (1897) Google Scholar), adipose tissue clearly exerts a major influence on carbohydrate metabolism because changes in body fat mass can have substantial effects on insulin action and glucose tolerance. Moreover, recent studies demonstrating that adipose tissue can secrete a number of molecules that modulate carbohydrate and lipid metabolism strongly suggest that body fat is more than just a passive reservoir for fuel in the form of triglycerides. Recently, a new hormone produced by fat cells and termed resistin was discovered that could represent an important link between obesity and insulin-resistant diabetes (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar, 3Holcomb I.N. Kabakoff R.C. Chan B. Baker T.W. Gurney A. Henzel W. Nelson C. Lowman H.B. Wright B.D. Skelton N.J. Frantz G.D. Tumas D.B. Peale Jr., F.V. Shelton D.L. Hebert C.C. EMBO J. 2000; 19: 4046-4055Crossref PubMed Scopus (600) Google Scholar, 4Kim K.H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). Resistin is a cysteine-rich polypeptide expressed primarily in white adipose tissue that is induced during 3T3-L1 adipogenesis and may also serve as a feedback regulator to inhibit adipocyte generation (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar, 3Holcomb I.N. Kabakoff R.C. Chan B. Baker T.W. Gurney A. Henzel W. Nelson C. Lowman H.B. Wright B.D. Skelton N.J. Frantz G.D. Tumas D.B. Peale Jr., F.V. Shelton D.L. Hebert C.C. EMBO J. 2000; 19: 4046-4055Crossref PubMed Scopus (600) Google Scholar, 4Kim K.H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). Message and protein levels of resistin are decreased by fasting and increased by refeeding, possibly in response to changes in insulin levels (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar, 4Kim K.H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). Moreover, Steppan et al. (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar) have found that treatment of mice with recombinant resistin can impair glucose tolerance and that administration of anti-resistin antibody improves blood glucose and insulin action in mice with diet-induced obesity (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar). Incubation of 3T3-L1 adipocytes with recombinant resistin has also been reported to inhibit insulin-stimulated glucose uptake (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar). In addition, resistin mRNA levels can be suppressed by exposure to either fatty acids or ligands for the peroxisome proliferator-activated receptor γ (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar). However, in various rodent models of obesity, conflicting results have been reported regarding the effects of systemically administered peroxisome proliferator-activated receptor γ ligands on resistin expression, perhaps because of the fact that these ligands can influence a host of factors that may differentially regulate resistin (5Banerjee R.R. Lazar M.A. J. Mol. Med. 2003; 81: 218-226Crossref PubMed Scopus (125) Google Scholar, 6Juan C.C. Au L.C. Fang V.S. Kang S.F. Ko Y.H. Kuo S.F. Hsu Y.P. Kwok C.F. Ho L.T. Biochem. Biophys. Res. Commun. 2001; 289: 1328-1333Crossref PubMed Scopus (114) Google Scholar, 7Moore G.B.T. Chapman H. Holder J.C. Lister C.A. Piercy V. Smith S.A. Clapham J.C. Biochem. Biophys. Res. Commun. 2001; 286: 735-741Crossref PubMed Scopus (141) Google Scholar, 8Way J.M. Gorgun C.Z. Tong Q. Uysal K.T. Brown K.K. Harrington W.W. Oliver Jr., W.R. Willson T.M. Kliewer S.A. Hotamisligil G.S. J. Biol. Chem. 2001; 27: 25651-25653Abstract Full Text Full Text PDF Scopus (406) Google Scholar). Based on measurements of resistin expression in fat tissue in humans and in animals with type 2 diabetes and or obesity, a number of investigators have recently raised questions regarding the potential relevance of resistin to the pathogenesis of insulin resistance. For example, whereas some investigators have found evidence of resistin expression in samples of human subcutaneous and abdominal fat (9McTernan C.L. McTernan P.G. Harte A.L. Levick P.L. Barnett A.H. Kumar S. Lancet. 2002; 359: 46-47Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 10Savage D.B. Sewter C.P. Klenk E.S. Segal D.G. Vidal-Puig A. Considine R.V. O'Rahilly S. Diabetes. 2001; 50: 2199-2202Crossref PubMed Scopus (704) Google Scholar), others have found it difficult to detect mRNA for resistin in either adipocytes or subcutaneous adipose tissue isolated from insulin-resistant subjects (11Nagaev I. Smith U. Biochem. Biophys. Res. Commun. 2001; 285: 561-564Crossref PubMed Scopus (370) Google Scholar). Expression of resistin in white adipose tissue has also been reported to be significantly decreased in several animal models of obesity-associated insulin resistance (6Juan C.C. Au L.C. Fang V.S. Kang S.F. Ko Y.H. Kuo S.F. Hsu Y.P. Kwok C.F. Ho L.T. Biochem. Biophys. Res. Commun. 2001; 289: 1328-1333Crossref PubMed Scopus (114) Google Scholar, 12Fukui Y. Motojima K. Diabetes Obes. Metab. 2002; 4: 342-345Crossref PubMed Scopus (53) Google Scholar, 13Fujita H. Fujishima H. Morii T. Koshimura J. Narita T. Kakei M. Ito S. Biochem. Biophys. Res. Commun. 2002; 298: 345-349Crossref PubMed Scopus (54) Google Scholar). However, the lack of correlation between resistin mRNA levels in isolated adipocytes and insulin resistance does not exclude the possibility that resistin may be contributing to the pathogenesis of disordered carbohydrate metabolism in either liver or skeletal muscle. In Sprague-Dawley rats, intra-arterial infusion of recombinant resistin over a period of 5 h has recently been reported to promote glucose intolerance by impairing insulin action on hepatic glucose metabolism (14Rajala M.W. Obici S. Scherer P.E. Rossetti L. J. Clin. Invest. 2003; 111: 225-230Crossref PubMed Scopus (485) Google Scholar). This observation raises the possibility that local secretion of resistin-like molecules into the portal venous circulation might play a role in the pathogenesis of type 2 diabetes. It is also conceivable that paracrine effects of resistin produced by adipocytes embedded deep within skeletal muscle might contribute to the pathogenesis of impaired glucose tolerance in the absence of changes in either circulating levels of resistin or resistin expression in visceral or superficial subcutaneous fat. However, few studies have been performed to directly investigate the effects of resistin on skeletal muscle glucose metabolism or the relationship between circulating levels of resistin and glucose tolerance. In this study, we investigated the chronic effects of resistin on glucose metabolism in the spontaneously hypertensive rat (SHR), 1The abbreviations used are: SHR, spontaneously hypertensive rat; NEFA, non-esterified fatty acid; OGTT, oral glucose tolerance test. a widely studied animal model of the hypertension metabolic syndrome that is predisposed to insulin resistance due at least in part to a genetic defect in the CD36 fatty acid transporter (15Pravenec M. Zídek V. Šimáková M. Kren V. Krenová D. Horký K. Jáchymová M. Míková B. Kazdová L. Aitman T.J. Churchill P.C. Webb R.C. Hingarh N.H. Yang Y. Wang J. St. Lezin E.M. Kurtz T.W. J. Clin. Invest. 1999; 103: 1651-1657Crossref PubMed Scopus (103) Google Scholar, 16Aitman T.J. Glazier A.M. Wallace C.A. Cooper L.D. Norsworthy P.J. Wahid F.N. Al-Majali K.M. Trembling P.M. Mann C.J. Shoulders C.C. Graf D. St. Lezin E. Kurtz T.W. Kren V. Pravenec M. Ibrahimi A. Abumrad N.A. Stanton L.W. Scott J. Nat. Genet. 1999; 21: 76-83Crossref PubMed Scopus (645) Google Scholar, 17Pravenec M. Landa V. Zídek V. Musilová A. Kren V. Kazdová L. Aitman T.J. Glazier A.M. Ibrahimi A. Abumrad N.A. Qi N. Wang J. St. Lezin E.M. Kurtz T.W. Nat. Genet. 2001; 27: 156-158Crossref PubMed Scopus (163) Google Scholar). We have found that transgenic expression of the mouse resistin gene under the control of the aP2 promoter in the SHR induces dyslipidema and increased muscle triglycerides, impairs oxidative and non-oxidative glucose disposal in skeletal muscle, and promotes glucose intolerance in the absence of detectable changes in circulating levels of resistin or insulin. In addition, we observed that recombinant resistin can inhibit glucose oxidation and insulin-stimulated glycogenesis in freshly isolated soleus muscle from non-transgenic SHR. These findings raise the possibility that paracrine actions of resistin secreted by adipocytes embedded within skeletal muscle or autocrine effects of resistin in adipocytes leading to the release of other prodiabetic effector molecules from fat or both may contribute to the pathogenesis of disordered skeletal muscle glucose metabolism and impaired glucose tolerance. Animals—The resistin transgene was expressed on the genetic background of the SHR/Ola strain (15Pravenec M. Zídek V. Šimáková M. Kren V. Krenová D. Horký K. Jáchymová M. Míková B. Kazdová L. Aitman T.J. Churchill P.C. Webb R.C. Hingarh N.H. Yang Y. Wang J. St. Lezin E.M. Kurtz T.W. J. Clin. Invest. 1999; 103: 1651-1657Crossref PubMed Scopus (103) Google Scholar, 16Aitman T.J. Glazier A.M. Wallace C.A. Cooper L.D. Norsworthy P.J. Wahid F.N. Al-Majali K.M. Trembling P.M. Mann C.J. Shoulders C.C. Graf D. St. Lezin E. Kurtz T.W. Kren V. Pravenec M. Ibrahimi A. Abumrad N.A. Stanton L.W. Scott J. Nat. Genet. 1999; 21: 76-83Crossref PubMed Scopus (645) Google Scholar, 17Pravenec M. Landa V. Zídek V. Musilová A. Kren V. Kazdová L. Aitman T.J. Glazier A.M. Ibrahimi A. Abumrad N.A. Qi N. Wang J. St. Lezin E.M. Kurtz T.W. Nat. Genet. 2001; 27: 156-158Crossref PubMed Scopus (163) Google Scholar). The rats were housed in an air-conditioned animal facility and allowed free access to food and water. Metabolic phenotypes were assessed in male control SHR (n = 10) and male resistin-transgenic SHR (n = 10) after the rats were fed a diet with 60% fructose (K4102.0 diet, Hope Farms, Woerden, The Netherlands) from the age of 8 weeks for 15 days. All of the experiments were performed in agreement with the Animal Protection Law of the Czech Republic (311/1997) and were approved by the Ethics Committee of the Institute of Physiology, Czech Academy of Sciences (Prague, Czech Republic). Transgenic Strain Derivation—Transgenic SHR were derived by microinjection of zygotes with a mouse resistin cDNA construct that was prepared by reverse transcriptase PCR of RNA from fat tissue of a BALB/c mouse. Resistin primers were designed according to the published resistin sequence (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar). The construct contained, in addition to cDNA of the mouse resistin gene, rabbit β-globin intron 2, a growth hormone poly(A) signal and the fat-specific aP2 promoter vector (kindly provided by Dr. Farid Chehab, University of California, San Francisco, CA). Microinjections of recombinant DNA into one or both pro-nuclei of fertilized ova were done according to Charreau et al. (18Charreau B. Menoret S. Tesson L. Soulillou J.-P. Anegon I. Rat Genome. 1997; 3: 125-132Google Scholar). Transgenic rats were detected by PCR using primers specific for the mouse resistin gene: upstream, 5′-tca aca aga agg agc tgt gg-3′, and downstream, 5′-cca gcc tgt ttt gtt tta ttt-′3. The official designation of the new resistin-transgenic strain is SHR/Ola-TgN(Retn)201Ipcv (abbreviated herein as the SHR-TG strain). Gene Expression Analysis of Transgenic SHR Rats—Northern blot analysis was used to confirm expression of the mouse resistin transgene and endogenous rat resistin gene in adipose tissue. The probe for Northern analysis was prepared by random primer labeling of the first 580 bp of the mouse resistin gene cut and purified from the transgene construct. Real-time PCR analysis was used to test for possible expression of the mouse resistin transgene in skeletal muscle. The cyclophilin (peptidylprolyl isomerase A) gene was used as an internal control with expression of the mouse resistin transgene relative to cyclophilin being determined in triplicate using the preferred method of Muller et al. (19Muller P.Y. Janovjak H. Miserez A.R. Dobbie Z. BioTechniques. 2002; 32: 1372-1379PubMed Google Scholar, 20Muller P.Y. Janovjak H. Miserez A.R. Dobbie Z. BioTechniques. 2002; 33: 514Google Scholar). The cDNA was prepared by reverse transcription of soleus muscle mRNA using random primers followed by real-time PCR amplification using QuantiTect SYBR Green reagents (Qiagen, Inc., Valencia, CA) on an Opticon continuous fluorescence detector (MJ Research, Waltham, MA). The upstream primers were 5′-caa atg ctg gac cca aca ca-3′ (cyclophilin A) and 5′-aga agg cac agc agt ctt-3′ (mouse resistin). The downstream primers were 5′-tgc cat cca acc act cag tc-3′ (cyclophilin A) and 5′-tgt cca gtc tat cct tgc a-3′ (mouse resistin). Western Blot Analysis—Proteins were extracted from 2 g of epididymal fat. Fat tissue was homogenized with 4 ml of ice-cold extracting buffer (0.3 m Tris-Cl, 0.14 m NaCl, 0.03 m KCl, 1% (w/v) SDS, 1% (v/v) Tween 20, pH 7.4) and 80 μl of protease inhibitors (protease inhibitor mixture for mammalian tissues, Sigma) for 1 min on ice. The samples then were agitated for 1 min by vortex and incubated for 10 min at 37 °C. Afterward, the samples were centrifuged for 10 min, 5300 × g, at 4 °C. The water phase was used as a protein extract. For SDS electrophoresis, the protein extracts were agitated with 2 volumes of sample buffer containing β-mercaptoethanol (Laemmli buffer system according to Mini-PROTEAN 3 cell instruction manual from Bio-Rad) and heated to 95 °C for 5 min. Samples were loaded on polyacrylamide gel (4% stacking gel and 13% resolving gel), and the electrophoresis was run at 70 V. The load of samples was 30 μl. For Western blotting, a wet protein transfer was done using Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) with transfer buffer (25 mm Tris, 192 mm glycine, 20% v/v methanol, 0.5% w/v SDS, pH 8.3) and nitrocellulose membrane (pore size 0.2 μm, Bio-Rad). Conditions were 30 V for 16 h at 4 °C. The membrane was blocked in 0.01 m phosphate-buffered saline, pH 7.4 (Sigma), containing 3% nonfat dry milk (Sigma) for 30 min at room temperature. Resistin protein was detected with a rabbit anti-mouse resistin antibody (Alpha Diagnostic International, Inc., San Antonio, TX). This antibody does not distinguish between mouse and rat resistin. Horseradish peroxidase-labeled donkey anti-rabbit IgG antibody was used as a secondary antibody. Primary and secondary antibodies were diluted in 0.01 m phosphate-buffered saline containing 1.5% nonfat dry milk. The incubation with the primary antibody was overnight at 4 °C and with the conjugate for 1 h at room temperature. The membrane then was treated with the ECL (Amersham Biosciences), and the signal was detected using the Luminiscent Image analysis system (LAS-1000+, Fuji) and quantified by AIDA image analyzer program (Raytest). Oral Glucose Tolerance Testing—Oral glucose tolerance tests (OGTT) were performed using a glucose load of 300 mg/100 g body weight after 7 h of fasting. Blood was drawn from the tail without anesthesia before the glucose load (0-min time point) and at 30, 60, and 120 min thereafter. Skeletal Muscle Glycogen Synthesis and Glucose Oxidation—Glycogen synthesis and glucose oxidation were determined in isolated soleus muscle by measuring the incorporation of [14C-U]glucose into glycogen and CO2 as described previously (21Vrána A. Poledne R. Fábry P. Kazdová L. Metabolism. 1978; 27: 885-888Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 22Qi N. Kazdová L. Zídek V. Landa V. Kren V. Pershadsingh H.A. St. Lezin E. Abumrad N.A. Pravenec M. Kurtz T.W. J. Biol. Chem. 2002; 277: 48501-48507Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The soleus muscles were attached to a stainless steel frame in situ at in vivo length by special clips and separated from other muscles and tendons and immediately incubated for 2 h in Krebs-Ringer bicarbonate buffer, pH 7.4, that contained 5.5 mm unlabeled glucose, 0.5 μCi/ml [14C-U]glucose, and 3 mg/ml bovine serum albumin (Armour, Fraction V) with or without 250 microunits/ml insulin. After a 2-h incubation, 0.3 ml of 1 m hyamine hydroxide was injected into central compartment of the incubation vessel and 0.5 ml of 1 m H2SO4 was injected into the main compartment to liberate CO2. The vessels were incubated for another 30 min, and the hyamine hydroxide was then quantitatively transferred into the scintillation vial containing 10 ml of toluene-based scintillation fluid for counting of radioactivity. For measurement of insulin-stimulated incorporation of glucose into glycogen, glycogen was extracted and glucose incorporation into glycogen was determined as described previously (21Vrána A. Poledne R. Fábry P. Kazdová L. Metabolism. 1978; 27: 885-888Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 22Qi N. Kazdová L. Zídek V. Landa V. Kren V. Pershadsingh H.A. St. Lezin E. Abumrad N.A. Pravenec M. Kurtz T.W. J. Biol. Chem. 2002; 277: 48501-48507Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Tissue Triglyceride Measurements—For determination of triglycerides in liver and soleus muscle, tissues were powdered under liquid N2 and extracted for 16 h in chloroform:methanol, after which 2% KH2PO4 was added and the solution was centrifuged. The organic phase was removed and evaporated under N2. The resulting pellet was dissolved in isopropyl alcohol, and triglyceride content was determined by enzymatic assay (Pliva-Lachema, Brno, Czech Republic). Effects of Recombinant Resistin on Insulin-stimulated Glucose Oxidation and Glycogen Synthesis in Skeletal Muscle Isolated from the SHR—Glycogenesis and glucose oxidation were measured as described above in soleus muscles isolated from 7-week-old male SHR/Ola (n = 5/each group) fed standard laboratory chow. Recombinant resistin (Alpha Diagnostic International, Inc.) was added to incubation media at a concentration of 600 ng/ml. Biochemical Analyses—Blood glucose levels were measured by the glucose oxidase assay (Pliva-Lachema) using tail vein blood drawn into 5% trichloroacetic acid and promptly centrifuged. Serum non-esterified fatty acid (NEFA) levels were determined using an acyl-CoA oxidase-based colorimetric kit (Roche Diagnostics). Serum triglyceride concentrations were measured by standard enzymatic methods (Pliva-Lachema). Serum insulin concentrations were determined using a rat insulin radioimmunoassay kit (Amersham Biosciences). Serum levels of leptin were determined using a rat leptin radioimmunoassay kit from Linco Research (St. Charles, MO). Serum resistin concentrations were determined at Linco Research, Inc. with an immunoassay that cross-reacts with mouse and rat resistin (Linco Research, Inc.). Statistical Analysis—All of the data are expressed as means ± S.E. Differences between control and experimental groups were evaluated by paired or non-paired Student's t tests as appropriate. Statistical significance was defined as p < 0.05. Transgenic Expression of Resistin—Transgene-positive SHR and transgene-negative controls were identified by genotyping offspring derived from crosses of a founder male with transgene-negative SHR females. In the epididymal fat from the SHR transgene-positive line, Northern blot analysis confirmed the expression of both the mouse resistin transgene and the endogenous rat resistin gene (Fig. 1a). The transgene-negative controls showed expression of only the endogenous rat resistin gene. Fig. 1a shows the presence of the 0.8- and 1.4-kb pair transcripts that are characteristic of endogenous rat resistin in both the transgene-positive SHR and in a transgene-negative control. These two transcripts are similar in size to those previously reported for the rat, which is known to express two resistin transcripts, whereas the mouse expresses only one resistin transcript (4Kim K.H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). In the SHR-transgenic line, a single transcript for the mouse resistin transgene could be detected in moderate amounts that is distinct in size from the endogenous rat resistin transcripts and that is slightly larger than the wild type mouse resistin transcript because of its longer 3′-untranslated tail. In addition to detecting mRNA for the mouse resistin transgene by Northern blot analysis in epididymal fat from the transgenic strain, we were able to detect low level skeletal muscle expression of the mouse resistin transgene relative to that of cyclophilin by real-time PCR (Fig. 1b). Transgene-negative controls showed no expression of the mouse resistin gene in fat or muscle when tested by either Northern blot analysis or real-time PCR. Western blot analysis of adipose tissue demonstrated greater expression of resistin protein in the transgene-positive rats than in transgene-negative controls (Fig. 1c). Effects of Resistin Transgene on Body Weight, Serum Phenotypes, and Tissue Lipid Levels—At the time of sacrifice, there were no significant differences between transgene-positive rats and transgene-negative controls with respect to either body weight or epididymal fat weight (Table I). Serum resistin levels were similar between SHR transgene-positive rats and transgene-negative controls as measured by an immunoassay that cross-reacts with mouse and rat resistin (Table I). Serum concentrations of insulin and glucose in transgene-positive rats, 1.2 ± 0.10 nmol/liter and 6.7 ± 0.2 mmol/liter, were similar to those in transgene-negative controls, 1.2 ± 0.13 nmol/liter and 6.8 ± 0. 2 mmol/liter, respectively. In transgene-positive rats, serum levels of leptin, 3.8 ± 0.8 ng/ml, were also not different compared with those in transgene negative controls, 3.9 ± 0.7 ng/ml. In contrast, serum concentrations of NEFA were significantly increased in transgenic animals compared with controls (Fig. 2a). Increased serum NEFA levels were also observed in the transgene-positive rats during the oral glucose tolerance test (Fig. 2a). In the transgene-positive rats, the increased serum NEFA levels were associated with increased muscle triglycerides (Fig. 2b), whereas no differences in hepatic triglycerides were observed between the transgene-positive rats (16.4 ± 0.7 μmol/g) and the transgene-negative controls (16.4 ± 0.6 μmol/g).Table IBody weights, relative epididymal fat weights, and serum resistin concentrationsStrainBody weightEpididymal fat weightSerum resistingg/100 g body weightng/mlSHR control280 ± 31.05 ± 0.0521.8 ± 2.6SHR-TG278 ± 61.05 ± 0.0621.5 ± 2.4 Open table in a new tab Effects of Resistin Transgene on Oral Glucose Tolerance— The resistin-transgenic rats displayed impaired oral glucose tolerance (Fig. 3) with the area under the glucose tolerance curve being significantly greater in the transgene-positive line than in the transgene-negative controls (831 ± 13 mmol/2 h and 760 ± 12 mmol/2 h, respectively; p < 0.005) (Fig. 3). These results are consistent with the studies of Steppan et al. (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar) in which systemic administration of recombinant resistin was found to impair oral glucose tolerance in C57BL/6J mice. However, in contrast to the studies of Steppan et al. (2Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar) in which impaired oral glucose tolerance induced by injection of recombinant resistin was associated with increased serum levels of resistin, the current studies demonstrate that transgenic expression of mouse resistin on the SHR background can impair oral glucose tolerance in the absence of detectable changes in circulating resistin. Effects of Resistin Transgene on Skeletal Muscle Glucose Metabolism—In soleus muscle isolated from transgenic SHR expressing the mouse resistin gene, glycogenesis and glucose oxidation were significantly reduced in both the presence and absence of insulin (Fig. 4, a and b). Thus, both non-oxidative and oxidative glucose metabolism were impaired in skeletal muscle of transgenic rats compared with controls. These findings are in accord with the recent studies of Moon et al. (23Moon B. Kwan J.J. Duddy N. Sweeney G. Begum N. Am. J. Physiol. 2003; 285: E106-E115Crossref PubMed Scopus (145) Google Scholar) in which recombinant resistin was found to inhibit glucose uptake in cultured L6 skeletal muscle cells in both the presence and absence of insulin. However, in the studies of Moon et al. (23Moon B. Kwan J.J. Duddy N. Sweeney G. Begum N. Am. J. Physiol. 2003; 285: E106-E115Crossref PubMed Scopus (145) Google Scholar), the effects of recombinant resistin on metabolic pathways of glucose disposal were not investigated and the extent to which the effects of recombinant resistin on glucose metabolism in L6 cells resemble those in skeletal muscle tissue is unknown. The current findings indicate that transgenic expression of resistin can impair both of the main pathways that mediate skeletal muscle glucose disposal even in isolated tissue that has not been previously exposed to" @default.
• W2072676249 created "2016-06-24" @default.
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• W2072676249 date "2003-11-01" @default.
• W2072676249 modified "2023-10-12" @default.
• W2072676249 title "Transgenic and Recombinant Resistin Impair Skeletal Muscle Glucose Metabolism in the Spontaneously Hypertensive Rat" @default.
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