AKT phosphorylation is essential for insulin-induced relaxation of rat vascular smooth muscle cells.

Insulin resistance, a major factor in the development of type 2 diabetes, is known to be associated with defects in blood vessel relaxation. The role of Akt on insulin-induced relaxation of vascular smooth muscle cell (VSMC) was investigated using siRNA targeting Akt (siAKTc) and adenovirus constructing myristilated Akt to either suppress endogenous Akt or overexpress constitutively active Akt, respectively. siAKTc decreased both basal and insulin-induced phosphorylations of Akt and glycogen synthase kinase 3beta, abolishing insulin-induced nitric oxide synthase (iNOS) expression. cGMP-dependent kinase 1alpha (cGK1alpha) and myosin-bound phosphatase (MBP) activities, both downstream of iNOS, were also decreased. siAKTc treatment resulted in increased insulin and ANG II-stimulated phosphorylation of contractile apparatus, such as MBP substrate (MYPT1) and myosin light chain (MLC20), accompanied by increased Rho-associated kinase alpha (ROKalpha) activity, demonstrating the requirement of Akt for insulin-induced vasorelaxation. Corroborating these results, constitutively active Akt upregulated the signaling molecules involved in insulin-induced relaxation such as iNOS, cGK1alpha, and MBP activity, even in the absence of insulin stimulation. On the contrary, the contractile response involving the phosphorylation of MYPT1 and MLC20, and increased ROKalpha activity stimulated by ANG II were all abolished by overexpressing active Akt. In conclusion, we demonstrated here that insulin-induced VSMC relaxation is dependent on Akt activation via iNOS, cGK1alpha, and MBP activation, as well as the decreased phosphorylations of MYPT1 and MLC20 and decreased ROKalpha activity.

[1]  B. Fleshler,et al.  Physiology of the Gastrointestinal Tract , 1969 .

[2]  P. Cohen.  [26] Phosphorylase kinase from rabbit skeletal muscle , 1983 .

[3]  D. Hartshorne,et al.  Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. , 1985, The Journal of biological chemistry.

[4]  D. Hartshorne,et al.  Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin. , 1986, The Journal of biological chemistry.

[5]  P. Ganz,et al.  Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. , 1986, The New England journal of medicine.

[6]  D. Hartshorne,et al.  Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. , 1989, Biochemical and biophysical research communications.

[7]  M. Laakso,et al.  Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man. A novel mechanism for insulin resistance. , 1990, The Journal of clinical investigation.

[8]  C. Kahn,et al.  Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein , 1991, Nature.

[9]  M. Laakso,et al.  Impaired Insulin-Mediated Skeletal Muscle Blood Flow in Patients With NIDDM , 1992, Diabetes.

[10]  P. Cohen,et al.  Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. , 1992, The Journal of biological chemistry.

[11]  M. Tuck,et al.  Insulin attenuates agonist-mediated calcium mobilization in cultured rat vascular smooth muscle cells. , 1993, The Journal of clinical investigation.

[12]  A. Mark,et al.  The vasodilator action of insulin. Implications for the insulin hypothesis of hypertension. , 1993, Hypertension.

[13]  M. Creager,et al.  Impaired Endothelium‐Dependent Vasodilation in Patients With Insulin‐Dependent Diabetes Mellitus , 1993, Circulation.

[14]  M. Creager,et al.  IMPAIRED ENDOTHELIUMDEPENDENT VASODILATION IN PATIENTS WITH INSULINDEPENDENT DIABETES MELLITUS , 1993 .

[15]  L. Johnson,et al.  Physiology of the gastrointestinal tract , 2012 .

[16]  N. Begum,et al.  Regulation of protein phosphatase 1 and 2A activities by insulin during myogenesis in rat skeletal muscle cells in culture. , 1994, The Journal of biological chemistry.

[17]  J. Herrera Acosta.  [Insulin resistance and hypertension]. , 1994, Gaceta medica de Mexico.

[18]  P. Cohen,et al.  Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B , 1995, Nature.

[19]  William Arbuthnot Sir Lane,et al.  Role of IRS-2 in insulin and cytokine signalling , 1995, Nature.

[20]  B. Burgering,et al.  Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction , 1995, Nature.

[21]  V. Schini-Kerth,et al.  Nitric oxide synthases in vascular cells , 1995, Experimental physiology.

[22]  C. Seidel,et al.  Insulin-stimulated glucose transport inhibits Ca2+ influx and contraction in vascular smooth muscle. , 1995, Circulation.

[23]  P. Kuo,et al.  The Emerging Multifaceted Roles of Nitric Oxide , 1995, Annals of surgery.

[24]  S. Narumiya,et al.  Roles of insulin, guanosine 5'-[gamma-thio]triphosphate and phorbol 12-myristate 13-acetate in signalling pathways of GLUT4 translocation. , 1996, The Biochemical journal.

[25]  D. Hartshorne,et al.  Interactions and properties of smooth muscle myosin phosphatase. , 1996, Biochemistry.

[26]  Kozo Kaibuchi,et al.  Regulation of Myosin Phosphatase by Rho and Rho-Associated Kinase (Rho-Kinase) , 1996, Science.

[27]  N. van den Berghe,et al.  Clostridium botulinum C3 exoenzyme stimulates GLUT4-mediated glucose transport, but not glycogen synthesis, in 3T3-L1 adipocytes--a potential role of rho? , 1996, Biochemical and biophysical research communications.

[28]  C. Kahn,et al.  Cross-talk between the insulin and angiotensin signaling systems. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[29]  K. Kaibuchi,et al.  Rho-associated Kinase Directly Induces Smooth Muscle Contraction through Myosin Light Chain Phosphorylation* , 1997, The Journal of Biological Chemistry.

[30]  E. Van Obberghen,et al.  cAMP stimulates protein kinase B in a Wortmannin‐insensitive manner , 1997, FEBS letters.

[31]  M. Eto,et al.  Molecular cloning of a novel phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (CPI17) in smooth muscle: its specific localization in smooth muscle 1 , 1997 .

[32]  C. Kahn,et al.  Angiotensin II inhibits insulin signaling in aortic smooth muscle cells at multiple levels. A potential role for serine phosphorylation in insulin/angiotensin II crosstalk. , 1997, The Journal of clinical investigation.

[33]  P. Cohen,et al.  Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα , 1997, Current Biology.

[34]  J. Zierath,et al.  Improved Glucose Tolerance Restores Insulin-Stimulated Akt Kinase Activity and Glucose Transport in Skeletal Muscle From Diabetic Goto-Kakizaki Rats , 1997, Diabetes.

[35]  M. Eto,et al.  Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle. , 1997, FEBS letters.

[36]  R. Farese,et al.  Activation and Translocation of Rho (and ADP Ribosylation Factor) by Insulin in Rat Adipocytes , 1997, The Journal of Biological Chemistry.

[37]  C. Seidel,et al.  Insulin acutely inhibits cultured vascular smooth muscle cell contraction by a nitric oxide synthase-dependent pathway. , 1997, Hypertension.

[38]  T. Soderling,et al.  Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway , 1998, Nature.

[39]  Frank McCormick,et al.  Akt activation by growth factors is a multiple-step process: the role of the PH domain , 1998, Oncogene.

[40]  U. Laufs,et al.  Post-transcriptional Regulation of Endothelial Nitric Oxide Synthase mRNA Stability by Rho GTPase* , 1998, The Journal of Biological Chemistry.

[41]  M. Roussel,et al.  Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. , 1998, Genes & development.

[42]  Philip R. Cohen,et al.  Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. , 1998, Science.

[43]  L. Ragolia,et al.  Regulation of Mitogen-activated Protein Kinase Phosphatase-1 Induction by Insulin in Vascular Smooth Muscle Cells , 1998, The Journal of Biological Chemistry.

[44]  R. Farese,et al.  Comparative Effects of GTPγS and Insulin on the Activation of Rho, Phosphatidylinositol 3-Kinase, and Protein Kinase N in Rat Adipocytes , 1998, The Journal of Biological Chemistry.

[45]  M. Zvelebil,et al.  Human Phosphoinositide 3-Kinase C2β, the Role of Calcium and the C2 Domain in Enzyme Activity* , 1998, The Journal of Biological Chemistry.

[46]  J. Zierath,et al.  Insulin-Stimulated Akt Kinase Activity Is Reduced in Skeletal Muscle From NIDDM Subjects , 1998, Diabetes.

[47]  D. Hartshorne,et al.  Rho-associated Kinase of Chicken Gizzard Smooth Muscle* , 1999, The Journal of Biological Chemistry.

[48]  H. Ueno,et al.  Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt. , 1999, The Journal of biological chemistry.

[49]  D. Hartshorne,et al.  Inhibitory Phosphorylation Site for Rho-associated Kinase on Smooth Muscle Myosin Phosphatase* , 1999, The Journal of Biological Chemistry.

[50]  H. Ueno,et al.  Intracellular Signaling of Angiotensin II-induced p70 S6 Kinase Phosphorylation at Ser411 in Vascular Smooth Muscle Cells , 1999, The Journal of Biological Chemistry.

[51]  M. Horiuchi,et al.  Recent progress in angiotensin II type 2 receptor research in the cardiovascular system. , 1999, Hypertension.

[52]  T. Lincoln,et al.  Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha. , 1999, Science.

[53]  Fumio Matsumura,et al.  Distinct Roles of Rock (Rho-Kinase) and Mlck in Spatial Regulation of Mlc Phosphorylation for Assembly of Stress Fibers and Focal Adhesions in 3t3 Fibroblasts , 2000, The Journal of cell biology.

[54]  Haibin Xia,et al.  A simple method for the rapid generation of recombinant adenovirus vectors , 2000, Gene Therapy.

[55]  D. Wink,et al.  Effects of oxidative and nitrosative stress in cytotoxicity. , 2000, Seminars in perinatology.

[56]  L. Ragolia,et al.  Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways. , 2000, Molecular endocrinology.

[57]  R. D. Rudic,et al.  Acute modulation of endothelial Akt/PKB activity alters nitric oxide-dependent vasomotor activity in vivo. , 2000, The Journal of clinical investigation.

[58]  O. Sandu,et al.  Signal Transduction in Smooth Muscle Selected Contribution : Insulin utilizes NO / cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle , 2001 .

[59]  O. Sandu,et al.  Selected contribution: insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle. , 2001, Journal of applied physiology.

[60]  M. Morissette,et al.  Increased Expression and Activity of RhoA Are Associated With Increased DNA Synthesis and Reduced p27Kip1 Expression in the Vasculature of Hypertensive Rats , 2001, Circulation research.

[61]  P. L. Becker,et al.  Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells. , 2001, Arteriosclerosis, thrombosis, and vascular biology.

[62]  H. Abboud,et al.  Activation of renal signaling pathways in db/db mice with type 2 diabetes. , 2001, Kidney international.

[63]  O. Sandu,et al.  Negative regulation of rho signaling by insulin and its impact on actin cytoskeleton organization in vascular smooth muscle cells: role of nitric oxide and cyclic guanosine monophosphate signaling pathways. , 2002, Diabetes.

[64]  S. Cook,et al.  Phenotypic Spectrum Caused by Transgenic Overexpression of Activated Akt in the Heart* , 2002, The Journal of Biological Chemistry.

[65]  S. Lohmann,et al.  Active Rho Kinase (ROK-α) Associates with Insulin Receptor Substrate-1 and Inhibits Insulin Signaling in Vascular Smooth Muscle Cells* , 2002, The Journal of Biological Chemistry.

[66]  Jian-Mei Li,et al.  Angiotensin II subtype 2 receptor activation inhibits insulin-induced phosphoinositide 3-kinase and Akt and induces apoptosis in PC12W cells. , 2002, Molecular endocrinology.

[67]  J. Ross,et al.  Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[68]  David P. Wilson,et al.  The role of RhoA and Rho-associated kinase in vascular smooth muscle contraction , 2003, Current hypertension reports.

[69]  L. Cantley,et al.  Use of RNA Interference-mediated Gene Silencing and Adenoviral Overexpression to Elucidate the Roles of AKT/Protein Kinase B Isoforms in Insulin Actions* , 2003, Journal of Biological Chemistry.

[70]  J. Stock,et al.  Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta. , 2003, The Journal of clinical investigation.

[71]  R. Alexander,et al.  Role of p38 MAPK and MAPKAPK-2 in angiotensin II-induced Akt activation in vascular smooth muscle cells. , 2004, American journal of physiology. Cell physiology.

[72]  S. Lohmann,et al.  MKP-1 expression and stabilization and cGK Ialpha prevent diabetes- associated abnormalities in VSMC migration. , 2004, American journal of physiology. Cell physiology.

[73]  G. Sesti,et al.  Angiotensin II Impairs the Insulin Signaling Pathway Promoting Production of Nitric Oxide by Inducing Phosphorylation of Insulin Receptor Substrate-1 on Ser312 and Ser616 in Human Umbilical Vein Endothelial Cells , 2004, Circulation research.

[74]  Jian-Mei Li,et al.  Angiotensin II Type-1 Receptor Blocker Valsartan Enhances Insulin Sensitivity in Skeletal Muscles of Diabetic Mice , 2004, Hypertension.

[75]  D. Hartshorne,et al.  Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase* , 2004, Journal of Biological Chemistry.

[76]  K. Malik,et al.  Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2(+)-dependent phospholipase A2. , 2005, American journal of physiology. Heart and circulatory physiology.

[77]  M. Sajan,et al.  Insulin-Sensitive Protein Kinases (Atypical Protein Kinase C and Protein Kinase B/Akt): Actions and Defects in Obesity and Type II Diabetes , 2005, Experimental biology and medicine.

[78]  Young-Bum Kim,et al.  Role of Rho-kinase in regulation of insulin action and glucose homeostasis. , 2005, Cell metabolism.

[79]  Sheila M. Thomas,et al.  Role of Phosphoinositide 3-Kinase Regulatory Isoforms in Development and Actin Rearrangement , 2005, Molecular and Cellular Biology.

[80]  K. Malik,et al.  Angiotensin II-Induced Akt Activation through the Epidermal Growth Factor Receptor in Vascular Smooth Muscle Cells Is Mediated by Phospholipid Metabolites Derived by Activation of Phospholipase D , 2005, Journal of Pharmacology and Experimental Therapeutics.

[81]  R. Alexander,et al.  Mechanisms of Reactive Oxygen Species–Dependent Downregulation of Insulin Receptor Substrate-1 by Angiotensin II , 2005, Arteriosclerosis, thrombosis, and vascular biology.

[82]  R. Komers,et al.  Emerging role of Akt kinase/protein kinase B signaling in pathophysiology of diabetes and its complications. , 2005, Physiological research.

[83]  R. Carey,et al.  Increased renal production of angiotensin II and thromboxane B2 in conscious diabetic rats. , 2005, American journal of hypertension.

[84]  K. Homma,et al.  Rho‐kinase as a molecular target for insulin resistance and hypertension , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[85]  J. Sowers,et al.  Role of phosphatidylinositol 3-kinase/Akt pathway in angiotensin II and insulin-like growth factor-1 modulation of nitric oxide synthase in vascular smooth muscle cells , 2002, Endocrine.