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• W2116717483 abstract "Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin β1 subunit in striated muscle results in a near complete loss of integrin β1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin β1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR·Rictor·LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin β1 signaling events in mediating cross-talk to that of insulin action. Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin β1 subunit in striated muscle results in a near complete loss of integrin β1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin β1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR·Rictor·LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin β1 signaling events in mediating cross-talk to that of insulin action. Integrin receptors are a large family of integral membrane proteins composed of a single α and β subunit assembled into a heterodimeric complex. There are 19 α and 8 β mammalian subunit isoforms that combine to form 25 distinct α,β heterodimeric receptors (1Humphries M.J. Biochem. Soc. Trans. 2000; 28: 311-339Crossref PubMed Google Scholar, 2Clemmons D.R. Maile L.A. Mol. Endocrinol. 2005; 19: 1-11Crossref PubMed Scopus (91) Google Scholar, 3Akiyama S.K. Hum. Cell. 1996; 9: 181-186PubMed Google Scholar, 4Kuppuswamy D. Circ. Res. 2002; 90: 1240-1242Crossref PubMed Scopus (24) Google Scholar, 5Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3809) Google Scholar). These receptors play multiple critical roles in conveying extracellular signals to intracellular responses (outside-in signaling) as well as altering extracellular matrix interactions based upon intracellular changes (inside-out signaling). Despite the large overall number of integrin receptor complexes, skeletal muscle integrin receptors are limited to seven α subunit subtypes (α1, α3, α4, α5, α6, α7, and αν subunits), all associated with the β1 integrin subunit (6Schwander M. Shirasaki R. Pfaff S.L. Muller U. J. Neurosci. 2004; 24: 8181-8191Crossref PubMed Scopus (52) Google Scholar, 7Schwander M. Leu M. Stumm M. Dorchies O.M. Ruegg U.T. Schittny J. Muller U. Dev. Cell. 2003; 4: 673-685Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Several studies have suggested an important cross-talk between extracellular matrix and insulin signaling. For example, engagement of β1 subunit containing integrin receptors was observed to increase insulin-stimulated insulin receptor substrate (IRS) 2The abbreviations used are: IRS, insulin receptor substrate; ILK, integrin-linked kinase; MCK, muscle creatine kinase; Itgβ1, integrin β1; KO, knockout; EU, euglycemic-hyperinsulinemic; PDK, phosphoinositide-dependent protein kinase; PI 3-kinase, phosphatidylinositol 3-kinase; GLUT4, glucose transporter isoform 4; GSK, glycogen synthase kinase. 2The abbreviations used are: IRS, insulin receptor substrate; ILK, integrin-linked kinase; MCK, muscle creatine kinase; Itgβ1, integrin β1; KO, knockout; EU, euglycemic-hyperinsulinemic; PDK, phosphoinositide-dependent protein kinase; PI 3-kinase, phosphatidylinositol 3-kinase; GLUT4, glucose transporter isoform 4; GSK, glycogen synthase kinase. phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation of protein kinase B/Akt (8Guilherme A. Czech M.P. J. Biol. Chem. 1998; 273: 33119-33122Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 9King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (385) Google Scholar, 10Yujiri T. Nawata R. Takahashi T. Sato Y. Tanizawa Y. Kitamura T. Oka Y. J. Biol. Chem. 2003; 278: 3846-3851Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 11Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (945) Google Scholar). Integrin receptor regulation of focal adhesion kinase was reported to modulate insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton organization in cultured hepatocytes and myoblasts (12Huang D. Khoe M. Ilic D. Bryer-Ash M. Endocrinology. 2006; 147: 3333-3343Crossref PubMed Scopus (38) Google Scholar, 13Huang D. Cheung A.T. Parsons J.T. Bryer-Ash M. J. Biol. Chem. 2002; 277: 18151-18160Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Similarly, the integrin-linked kinase (ILK) was suggested to function as one of several potential upstream kinases that phosphorylate and activate Akt (14Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (965) Google Scholar, 15Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar, 16Pasquet J.M. Noury M. Nurden A.T. Thromb. Haemostasis. 2002; 88: 115-122Crossref PubMed Scopus (61) Google Scholar, 17Yamaji S. Suzuki A. Kanamori H. Mishima W. Takabayashi M. Fujimaki K. Tomita N. Fujisawa S. Ohno S. Ishigatsubo Y. Biochem. Biophys. Res. Commun. 2002; 297: 1324-1331Crossref PubMed Scopus (27) Google Scholar, 18Yamaji S. Suzuki A. Sugiyama Y. Koide Y. Yoshida M. Kanamori H. Mohri H. Ohno S. Ishigatsubo Y. J. Cell Biol. 2001; 153: 1251-1264Crossref PubMed Scopus (169) Google Scholar). In this regard small interfering RNA gene silencing of ILK in fibroblasts and conditional ILK gene knockouts in macrophages resulted in a near complete inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation concomitant with an inhibition of Akt activity and phosphorylation of Akt downstream targets (19Troussard A.A. Mawji N.M. Ong C. Mui A. St-Arnaud R. Dedhar S. J. Biol. Chem. 2003; 278: 22374-22378Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2) has been identified in several other studies as the Akt Ser-473 kinase (20Pearce L.R. Huang X. Boudeau J. Pawlowski R. Wullschleger S. Deak M. Ibrahim A.F. Gourlay R. Magnuson M.A. Alessi D.R. Biochem. J. 2007; 405: 513-522Crossref PubMed Scopus (359) Google Scholar, 21Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar). In addition to Ser-473, Akt protein kinase activation also requires phosphorylation on threonine 308 Thr-30 by phosphoinositide-dependent protein kinase, PDK1 (22Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5223) Google Scholar, 23Scheid M.P. Marignani P.A. Woodgett J.R. Mol. Cell. Biol. 2002; 22: 6247-6260Crossref PubMed Scopus (270) Google Scholar, 24Dormond O. Madsen J.C. Briscoe D.M. J. Biol. Chem. 2007; 282: 23679-23686Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In vivo, skeletal muscle is the primary tissue responsible for postprandial (insulin-stimulated) glucose disposal that results from the activation of signaling pathways leading to the translocation of the insulin-responsive glucose transporter, GLUT4, from intracellular sites to the cell surface membranes (25Zierath J.R. Krook A. Wallberg-Henriksson H. Mol. Cell. Biochem. 1998; 182: 153-160Crossref PubMed Scopus (78) Google Scholar, 26Bjornholm M. Kawano Y. Lehtihet M. Zierath J.R. Diabetes. 1997; 46: 524-527Crossref PubMed Scopus (0) Google Scholar). Dysregulation of any step of this process in skeletal muscle results in a state of insulin resistance, thereby predisposing an individual for the development of diabetes (27Caro J.F. Sinha M.K. Raju S.M. Ittoop O. Pories W.J. Flickinger E.G. Meelheim D. Dohm G.L. J. Clin. Investig. 1987; 79: 1330-1337Crossref PubMed Scopus (301) Google Scholar, 28Goodyear L.J. Giorgino F. Sherman L.A. Carey J. Smith R.J. Dohm G.L. J. Clin. Investig. 1995; 95: 2195-2204Crossref PubMed Scopus (475) Google Scholar, 29Cusi K. Maezono K. Osman A. Pendergrass M. Patti M.E. Pratipanawatr T. DeFronzo R.A. Kahn C.R. Mandarino L.J. J. Clin. Investig. 2000; 105: 311-320Crossref PubMed Scopus (904) Google Scholar, 30Mandarino L.J. Consoli A. Jain A. Kelley D.E. Am. J. Physiol. 1996; 270: E463-E470PubMed Google Scholar, 31Damsbo P. Vaag A. Hother-Nielsen O. Beck-Nielsen H. Diabetologia. 1991; 34: 239-245Crossref PubMed Scopus (194) Google Scholar, 32Johnson A.B. Argyraki M. Thow J.C. Jones I.R. Broughton D. Miller M. Taylor R. Metabolism. 1991; 40: 252-260Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 33Kim Y.B. Nikoulina S.E. Ciaraldi T.P. Henry R.R. Kahn B.B. J. Clin. Investig. 1999; 104: 733-741Crossref PubMed Scopus (366) Google Scholar). Although studies described above have utilized a variety of tissue culture cell systems to address the potential involvement of integrin receptor signaling in insulin action, to date there has not been any investigation of integrin function on insulin action or glucose homeostasis in vivo. To address this issue, we have taken advantage of Cre-LoxP technology to inactivate the β1 integrin receptor subunit gene in striated muscle. We have observed that muscle creatine kinase-specific integrin β1 knock-out (MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose infusion rate and glucose clearance. The impairment of insulin-stimulated skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK expression. Together, these data demonstrate an important cross-talk between integrin receptor function and insulin action and suggests that ILK may function as an Akt Ser-473 kinase in skeletal muscle. Muscle-specific β1 Integrin Subunit Knock-out Mice—The floxed β1 integrin subunit (Itgβ1) mice were generated as previously described (34Potocnik A.J. Brakebusch C. Fassler R. Immunity. 2000; 12: 653-663Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). To obtain striated muscle-specific knockouts, the floxed Itgβ1 mice were mated with the MCK-Cre recombinase transgenic mice (35Bruning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar). All the mice strains used in this study were backcrossed 6-9 generations into the C57Bl6/J strain. Genotyping were performed by PCR using genomic DNA isolated from the tails of 3-4-week-old mice. The primers (5′-TGATGAGGTTCGCAAGAACC-3′ and 5′-CCATGAGTGAACGAACCTGG-3′) for identifying carriers of the Cre-recombinase transgenic were used under the following conditions: 1 cycle of 94 °C for 5 min, 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min followed by 1 cycle of 72 °C for 10 min. The primers for the floxed Itgβ1 genotyping (5′-AGGTGCCCTTCCCTCTAGA-3′ and 5′-GTGAAGTAGGTGAAAGGTAAC) were used under the following conditions: 1 cycle of 94 °C for 2 min, 35 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min followed by 1 cycle of 72 °C for 10 min. Mice were housed in a temperature-controlled environment with a 12-h light/12-h dark cycle and provided a standard chow diet with free access to food and water. Food intake and weights were determined daily, and all studies were performed on 14-week-old male mice. All animal protocols were performed in accordance with Stony Brook University Institutional Animal Care and Use Committee approval. Plasma Analysis and Immunoblotting—Fasting blood samples were collected by tail bleeding after a 14-h fast. Glucose was measured using a glucose oxidase method on a Beckman glucose analyzer 2 (Beckman Instruments Inc., Fullerton, CA). Insulin levels were determined by enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden). For acute insulin stimulation, the mice were fasted for 14 h and given an intraperitoneal injection with 1 unit/kg human recombinant insulin (Lilly). Ten minutes later the animals were anesthetized with pentobarbital sodium (50 mg/kg), and tissue samples were collected and immediately frozen at -80 °C. Tissues isolated from both EU clamps and acute insulin stimulation were homogenized in ice-cold lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 0.25% sodium deoxycholate, 1 mm NaF, 1 mm Na3VO4, and 2 mm Na4P2O7) containing a protease inhibitor mixture (Roche Diagnostics). The resultant lysates were centrifuged at 16,000 × g for 60 min at 4 °C, and protein concentrations were quantified using the BCA (bicinchoninic acid) protein assays (Pierce). The protein samples (30 μg) were separated on a 4-12% gradient SDS-PAGE gel and transferred to nitrocellulose membranes using a semidry electroblotter (Owl Separation System, Portsmouth, NH). Membranes were immunoblotted with β1 integrin monoclonal antibody (BD Pharmingen), GLUT4 polyclonal antibody (East Acres Biologicals Inc.), phospho-Ser-473, and phospho-Thr-308 Akt monoclonal antibodies, pan-protein kinase B/Akt polyclonal antibody, phospho-Ser-9-GSK3β, total GSK3β, phospho-Thr-642-AS160, total AS160 (Cell Signaling Technology, Inc.), ILK and talin monoclonal antibodies (Sigma), and α-dystroglycan antibody as previously described (36Kanagawa M. Michele D.E. Satz J.S. Barresi R. Kusano H. Sasaki T. Timpl R. Henry M.D. Campbell K.P. FEBS Lett. 2005; 579: 4792-4796Crossref PubMed Scopus (43) Google Scholar). Phospho-IRS1 and p110 association was determined by PY20 (BD Biosciences) and p110 (R&D Systems) immunoblotting of IRS1 immunoprecipitates (BD Biosciences). Quantification of all immunoblots was performed using NIH IMAGE software. In Vivo Assessment of Insulin Action and Glucose Metabolism—Four days before the experiment, the mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and an indwelling catheter was introduced in the left internal jugular vein. The catheters were externalized through an incision in the skin flap behind the head, and the mice were returned to individual cages after the surgery. The mice were fully recovered from the surgery before the in vivo experiments, as reflected by their reaching preoperative weight. After an overnight fast, EU clamps were conducted in conscious mice as previously described (37Kim J.K. Wi J.K. Youn J.H. Diabetes. 1996; 45: 651-658Crossref PubMed Scopus (66) Google Scholar). The 2-h EU clamp was conducted with a primecontinuous infusion of human insulin (2.5 milliunits/kg/min) and a variable infusion of 20% glucose to maintain glucose at ∼110 mg/dl. Insulin-stimulated whole body glucose metabolism was estimated using a prime continuous infusion of [3-3H]glucose (10 μCi bolus, 0.1 μCi/min; PerkinElmer Life Sciences). To determine the rate of basal glucose turnover, [3-3H]glucose (0.05 μCi/min) was infused for 2 h (basal period) before starting the EU clamp, and a blood sample was taken at the end of this basal period. To assess insulin-stimulated tissue-specific glucose uptake, 2-deoxy-d-[1-14C]glucose (PerkinElmer Life Sciences) was administered as a bolus (10 μCi) 75 min after the start of the clamp. Blood samples were taken at 80, 85, 90, 100, 110, and 120 min after the start of the EU clamp. To estimate basal muscle glucose uptake, 2-deoxy-d-[1-14C]glucose was infused with isotonic saline. All infusions were performed using microdialysis pumps (CMA/Microdialysis). At the end of the EU clamp, animals were euthanized with pentobarbital sodium (50 mg/kg), and different muscle groups, adipose tissue, heart, and liver were rapidly dissected and frozen at -80 °C for analysis. During the clamp plasma glucose was monitored using 10 μl of plasma by glucose analyzer 2. For the determination of plasma [3-3H]glucose and 2-deoxy-d-[1-14C]glucose concentrations, plasma was deproteinized with ZnSO4 and Ba(OH)2, dried to remove 3H2O, resuspended in water, and counted in scintillation fluid (Ultima Gold; Packard Instrument Co.). The plasma concentration of 3H2O was determined by the difference between 3H counts without and with drying. For the determination of tissue 2-deoxy-d-[1-14C]glucose (2-DG)-6-phosphate (2-DG-6-P) content, tissue samples were homogenized, and the supernatants were subjected to an ion-exchange column to separate 2-DG-6-P from 2-DG, as described previously (38Ohshima K. Shargill N.S. Chan T.M. Bray G.A. Am. J. Physiol. 1984; 246: E193-E197Crossref PubMed Google Scholar). The radioactivity of 3H in tissue glycogen was determined by digesting tissue samples in KOH and precipitating glycogen with ethanol as previously described (37Kim J.K. Wi J.K. Youn J.H. Diabetes. 1996; 45: 651-658Crossref PubMed Scopus (66) Google Scholar). Muscle glycogen synthesis was calculated as muscle [3H]glycogen content divided by the area under the plasma [3H]glucose-specific activity profile. Muscle glycolysis was estimated as the difference between muscle glucose uptake and muscle glycogen synthesis. Activity and Exercise Capacity—Spontaneous locomoter activity of control Itgβ1flox/flox and MCKItgβ1 KO mice was determined by quantifying the number of beam breaks in xy axis of an Oxymax open-circuit indirect calorimetry system (Columbus Instruments, Columbus, OH). During the first 48 h of the experiment, animals were allowed free access to food and water. After 48 h, animals were still allowed free access to water, but they were not fed in the evening for 12 h (from 7 p.m. to 7 a.m.) and then re-fed for 24 h (7 a.m. to 7 a.m.). At the end of the experiment, the animals underwent an 8-h fast (7 a.m. to 3 p.m.). The locomotor activity was monitored by the number of beam breaks and was averaged and expressed as counts/h. Running capacity was determined as described by Koch and Britton (39Koch L.G. Britton S.L. Physiol. Genomics. 2001; 5: 45-52Crossref PubMed Scopus (280) Google Scholar). Briefly, the MCKItgβ1 KO and Itgβ1flox/flox mice were acclimatized to a treadmill (Columbus Instruments) by running at 10 m/min for 15 min over 3 consecutive days. On the fourth day, groups of mice were run at 10 m/min on a 0° grade or 10 m/min on a 15° grade incline. Immunofluorescence Microscopy—After an overnight fast (14 h), the mice were euthanized. The tissues were removed and embedded in optimal cutting temperature compound. The frozen tissue cross-sections (10 μm) were blocked with 3% bovine serum albumin in phosphate-buffered saline for 60 min at room temperature. Primary antibodies were used at the following dilutions: β1 integrin polyclonal antibody (1:100) and talin antibody (1:50). Fluorescently conjugated secondary antibodies (1:100, Jackson ImmunoResearch Laboratories) were added to the sections for 30 min at room temperature. Filamentous (F-) actin was visualized by incubation of the fixed tissue with phalloidin-fluorescein isothiocyanate (1:1000) for 30 min. After extensive washes with phosphate-buffered saline, the slides were mounted with Vectashield Mounting Medium (Vector Laboratories). The slides were observed with confocal fluorescent microscopy (model LSM510; Carl Zeiss MicroImaging, Inc.). Statistical Analysis—Results are represented as the means ± S.E. Statistical significance was determined using an unpaired two-tailed Student's t test, with p < 0.05 considered significant. Generation of Muscle-specific β1 Integrin Knock-out Mice—To investigate the selective function of the β1 integrin subunit on insulin action and glucose homeostasis, we generated a muscle-specific-deficient Itgβ1 mouse by crossing mice carrying a “floxed” allele of β1 integrin (Itgβ1) in which loxP sites were introduced flanking exon 2 of the entire mouse β1 integrin gene (40Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (443) Google Scholar, 41Brakebusch C. Grose R. Quondamatteo F. Ramirez A. Jorcano J.L. Pirro A. Svensson M. Herken R. Sasaki T. Timpl R. Werner S. Fassler R. EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (317) Google Scholar) with transgenic mice carrying the MCK-promoter driving Cre-recombinase (35Bruning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar). Breeding of Itgβ1flox/+ and MCK-Cre+/- mice resulted in double heterozygous animals that were then bred with Itgβ1flox/+ mice to obtain Itgβ1flox/flox:MCK-Cre offspring; that is, mice with a disruption of β1 integrin specifically in striated muscle (MCKItgβ1 KO). Offspring from these crosses resulted in the expected frequency of 12.5% for a trait requiring two independent loci, demonstrating the absence of embryonic lethality. To verify the extent of Itgβ1 recombination, we examined various tissues in two independent MCK-Cre-crossed mice by immunoblotting. These data demonstrated an almost complete loss of β1 integrin protein expression in skeletal and cardiac muscle with essentially wild type levels of protein expression in all the other tissues examined (Fig. 1). The p115 Golgi protein was used a protein loading control. General phenotypic characteristics of the MCKItgβ1 KO mice demonstrated that up to 14 weeks of age, there was no significant difference in overall body weight, food intake, fasting plasma glucose, and insulin concentrations compared with control Itgβ1flox/flox and wild type mice (Table 1).TABLE 1Physical parameters of MCKItgβ1 KO miceWild typeItgβp1flox/floxMCKItgβ1KOFood intake (g/day)3.19 ± 0.83.21 ± 0.63.16 ± 0.74Weight (g)23.8 ± 1.324.9 ± 1.522.7 ± 1.19Fasting glucose (mg/dl)118.6 ± 9.8112.6 ± 9.8105 ± 9.9Fasting insulin (milliunits/liter)12.95 ± 1.612.75 ± 1.113.08 ± 0.76 Open table in a new tab Loss of Integrin β1 Expression in Skeletal Muscle Is Associated with Reduced Expression Levels of Talin and Actin Disorganization—Morphological examination of control skeletal muscle by confocal immunofluorescence microscopy demonstrated the presence of the integrin β1 subunit in the sarcolemma membrane (Fig. 2, panel a). As expected, there was a dramatic loss of integrin β1 expression in skeletal muscle of the MCKItgβ1 KO mice (Fig. 2, panel b). A concomitant loss of expression of the integrin β1 interacting protein talin was also observed in skeletal muscles of MCKItgβ1 KO mice (Fig. 2, panels c and d). Because β1 integrin receptors associate with the actin cytoskeleton via talin, we next investigated F-actin expression using phalloidin-fluorescein isothiocyanate staining (Fig. 3A, panels a-f). Concomitant with the reduction in talin there was reduced expression (52%) of F-actin in skeletal muscle of the MCKItgβ1 KO mice (Fig. 3B). The reduction in both talin and F-actin in skeletal muscle of the MCKItgβ1 KO mice occurred without a significant change in α-dystroglycan expression (data not shown).FIGURE 3Loss of β1 integrin receptor subunit expression results in a partial reduction and disorganization of F-actin filaments. A, gastrocnemius muscle was isolated from 14-week-old Itgβ1flox/flox and littermate MCK-Itgβ1 KO mice. Sections were subjected to immunofluorescence analysis for β1 integrin expression (panels a and b) and F-actin (phalloidin-fluorescein isothiocyanate staining) levels (panels c and d). The merged images are shown in panels e and f. B, quantification of F-actin levels was determined by fluorescent intensity. n = 6-10 independent experiments. *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because there was significant morphological disorganization of muscle actin structure, we assessed muscle function by first assessing spontaneous motor activity (Fig. 4A). Over the first 48-h period, the mice were allowed to eat ad libitum, and there was no significant difference in activity during either the 12-h dark or 12-h light cycle. Similarly, locomotor activity was essentially identical when the animals were fasted either during the dark cycle or during the light cycle. To examine the ability of the mice to respond to a greater demand of motor activity, we next determined the exercise tolerance of the MCKItgβ1 KO mice (Fig. 4B). The control Itgβ1flox/flox and the MCKItgβ1 KO mice were run on a level treadmill (0° degree) at 10 m/min. Over the 25-min challenge the mice tested were able to maintain this pace, and no significant differences between the Itgβ1flox/flox control and MCK-Itgβ1 KO mice were observed. In contrast, on a 15° grade incline, on average the MCKItgβ1 KO mice could only run for 12 min, whereas the control mice were able to maintain this pace for more than 20 min. MCKItgβ1 KO Mice Display Insulin Resistance Due to Decreased Muscle Glucose Metabolism—To investigate the consequences of the muscle-specific ablation of the integrin β1 subunit on whole body glucose homeostasis and tissue-specific insulin action, we next examined changes in glucose and insulin levels during an intraperitoneal glucose tolerance test (Fig. 5). As reported in Table 1, there was no significant difference in the fasting plasma glucose levels between the controls and the KO mice. However the MCKItgβ1 KO mice displayed a trend toward impaired glucose tolerance that was significant at 120 min after glucose injection (Fig. 5A). Similarly, the fasting insulin levels were also not different compared with controls, but during the intraperitoneal glucose tolerance test the insulin levels were significantly elevated in the MCKItgβ1 KO mice (Fig. 5B). These data suggest that the MCKItgβ1 KO mice display peripheral tissue insulin resistance that is compensated for by increased beta cell insulin secretion to maintain euglycemia. To more directly determine whether these mice are in fact insulin-resistant, we next performed a 2-h EU clamp in conscious MCKItgβ1 KO and Itgβ1flox/flox mice. No differences in plasma glucose or insulin levels were observed in either the basal or during the euglycemic-hyperinsulinemic clamp state (Fig. 6, A and B). However during the EU clamp, the rate of glucose infusion needed to maintain euglycemia increased rapidly in the control mice and reached a steady state. In contrast, the glucose infusion rate in response to insulin was reduced by 44% in the MCKItgβ1 KO mice compared with the control Itgβ1flox/flox mice (0.20 ± 0.03 versus 0.36 ± 0.02 mmol/kg/min) (Fig. 7A). Although there was no significant difference in glucose clearance between the MCKItgβ1 KO and control mice in the basal state, insulin-stimulated glucose clearance was significant decrease by 48% in the MCKItgβ1 KO mice (70.5 ± 5.6 versus 36.8 ± 9.4 ml/kg/min) (Fig. 7B). These data directly demonstrate the presence of insulin resistance in the MCKItgβ1 KO.FIGURE 7Loss of skeletal muscleβ1 integrin receptor subunit expression results in a decreased rate of insulin-stimulated glucose infusion rate and glucose clearance. A, EU clamps were used to assess whole body insulin sensitivity by determining the glucose infusion rate required to maintain euglycemia into control Itgβ1flox/flox (open boxes) and MCKItgβ1KO (filled boxes) mice. B, glucose clearance was determined as [3-3H]glucose-specific activity trace infusion rate and weight of mice in the basal or EU clamp state. These data represent the means ± S.E. from 7-10 individual mice per group. *, p < 0.05.View Large" @default.
• W2116717483 created "2016-06-24" @default.
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• W2116717483 creator A5069706521 @default.
• W2116717483 creator A5074572564 @default.
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• W2116717483 date "2009-02-01" @default.
• W2116717483 modified "2023-09-30" @default.
• W2116717483 title "Insulin Resistance in Striated Muscle-specific Integrin Receptor β1-deficient Mice" @default.
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