Met³²⁶lle Aminoacid Polymorphism in the Human p85α Gene has no Major Impact on Early Insulin Signaling in Type 2 Diabetes

Abstract

Class Iα phosphatidylinositol (PI) 3-kinase is an important enzyme in the early insulin signaling cascade, and plays a key role in insulin-mediated glucose transport. Despite extensive investigation, the genes responsible for the development of the common forms of type 2 diabetes remain unknown. This study was performed to identify variants in the coding region of p85α, the regulatory subunit of PI 3-kinase. Fibroblasts from skin biopsies from type 2 diabetics and controls were established to address this issue. P85α cDNA was sequenced, and a single point mutation at codon 326 was found. This mutation resulted in a homozygous missense amino acid change Met → Ile in one subject with type 2 diabetes and heterozygous variant in two other diabetic patients and one with severe insulin resistance. Interestingly, those patients revealed an impaired insulin-mediated insulin receptor substrate (IRS)-1 binding to p85α without any alteration in IRS-2/p85α association. Furthermore, IRS-1, IRS-2, p85α and MAPK protein contents were not significantly changed, and neither were MAPK or Akt phosphorylation. We conclude from our data that this variant may have only minor impact on signaling events; however, in combination with variants in other genes encoding signaling proteins, this may have a functional impact on early insulin signaling.

Key words

PI 3-kinase - Mutation - Insulin action - Type 2 diabetes - SNP

References

• 1 DeFronzo R A. Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidaemia and atherosclerosis.  Neth J Med. 1997;  50 191-197
• 2 Newman B, Selby J V, King M C, Slemenda C, Fabsitz R, Friedman G D. Concordance for type 2 (non-insulin-dependent) diabetes mellitus in male twins.  Diabetologia. 1987;  30 763-768
• 3 Rich S S. Mapping genes in diabetes. Genetic epidemiological perspective.  Diabetes. 1990;  39 1315-1319
• 4 Lillioja S, Mott D M, Spraul M, Ferraro R, Foley J E, Ravussin E, Knowler W C, Bennett P H, Bogardus C. Insulin resistance and insulin secretory dysfunction as precursors of non-insulin-dependent diabetes mellitus. Prospective studies of Pima Indians.  N Engl J Med. 1993;  329 1988-1992
• 5 Martin B C, Warram J H, Krolewski A S, Bergman R N, Soeldner J S, Kahn C R. Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study [see comments].  Lancet. 1992;  340 925-929
• 6 Taylor S I, Cama A, Accili D, Barbetti F, Quon M J, de la Luz Sierra M, Suzuki Y, Koller E, Levy-Toledano R, Wertheimer E. et al . Mutations in the insulin receptor gene.  Endocr Rev. 1992;  13 566-595
• 7 Moller D E, Cohen O, Yamaguchi Y, Assiz R, Grigorescu F, Eberle A, Morrow L A, Moses A C, Flier J S. Prevalence of mutations in the insulin receptor gene in subjects with features of the type A syndrome of insulin resistance.  Diabetes. 1994;  43 247-255
• 8 Moller D E, Bjorbaek C, Vidal-Puig A. Candidate genes for insulin resistance.  Diabetes Care. 1996;  19 396-400
• 9 Almind K, Doria A, Kahn C R. Putting the genes for type II diabetes on the map.  Nat Med. 2001;  7 277-279
• 10 Sun X J, Rothenberg P, Kahn C R, Backer J M, Araki E, Wilden P A, Cahill D A, Goldstein B J, White M F. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.  Nature. 1991;  352 73-77
• 11 Sun X J, Wang L M, Zhang Y, Yenush L, Myers M G, Glasheen E, Lane W S, Pierce J H, White M F. Role of IRS-2 in insulin and cytokine signaling.  Nature. 1995;  377 173-177
• 12 Vanhaesebroeck B, Leevers S J, Panayotou G, Waterfield M D. Phosphoinositide 3-kinases: a conserved family of signal transducers.  Trends Biochem Sci. 1997;  22 267-272
• 13 Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin.  J Biol Chem. 1994;  269 3568-3573
• 14 Yeh J I, Gulve E A, Rameh L, Birnbaum M J. The effects of wortmannin on rat skeletal muscle. Dissociation of signaling pathways for insulin- and contraction-activated hexose transport.  J Biol Chem. 1995;  270 2107-2111
• 15 Shepherd P R, Withers D J, Siddle K. Phosphoinositide 3-kinase: the key switch mechanism in insulin signaling [published erratum appears in Biochem J 1998 Nov 1; 335 (Pt 3): 711].  Biochem J. 1998;  333 471-490
• 16 Escobedo J A, Navankasattusas S, Kavanaugh W M, Milfay D, Fried V A, Williams L T. cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor.  Cell. 1991;  65 75-82
• 17 Hu P, Margolis B, Skolnik E Y, Lammers R, Ullrich A, Schlessinger J. Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.  Mol Cell Biol. 1992;  12 981-990
• 18 Lamphere L, Carpenter C L, Sheng Z F, Kallen R G, Lienhard G E. Activation of PI 3-kinase in 3T3-L1 adipocytes by association with insulin receptor substrate-1.  Am J Physiol. 1994;  266 E486-494
• 19 Dhand R, Hiles I, Panayotou G, Roche S, Fry M J, Gout I, Totty N F, Truong O, Vicendo P, Yonezawa K. et al . PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.  Embo J. 1994;  13 522-533
• 20 Otsu M, Hiles I, Gout I, Fry M J, Ruiz-Larrea F, Panayotou G, Thompson A, Dhand R, Hsuan J, Totty N. et al . Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase.  Cell. 1991;  65 91-104
• 21 Antonetti D A, Algenstaedt P, Kahn C R. Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.  Mol Cell Biol. 1996;  16 2195-2203
• 22 Algenstaedt P, Antonetti D A, Yaffe M B, Kahn C R. Insulin receptor substrate proteins create a link between the tyrosine phosphorylation cascade and the Ca2+-ATPases in muscle and heart.  J Biol Chem. 1997;  272 23 696-23 702
• 23 Fruman D A, Cantley L C, Carpenter C L. Structural organization and alternative splicing of the murine phosphoinositide 3-kinase p85 alpha gene.  Genomics. 1996;  37 113-121
• 24 Inukai K, Anai M, van Breda E, Hosaka T, Katagiri H, Funaki M, Fukushima Y, Ogihara T, Yazaki Y, Kikuchi M, Oka Y, Asano T. A novel 55-kDa regulatory subunit for phosphatidylinositol 3-kinase structurally similar to p55PIK Is generated by alternative splicing of the p85alpha gene.  J Biol Chem. 1996;  271 5317-5320
• 25 Pons S, Asano T, Glasheen E, Miralpeix M, Zhang Y, Fisher T L, Myers M G Jr, Sun X J, White M. F. The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase.  Mol Cell Biol. 1995;  15 4453-4465
• 26 Flier J S, Young J B, Landsberg L. Familial insulin resistance with acanthosis nigricans, acral hypertrophy, and muscle cramps.  N Engl J Med. 1980;  303 970-973
• 27 Dib K, Whitehead J P, Humphreys P J, Soos M A, Baynes K C, Kumar S, Harvey T, O’Rahilly S. Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. A disorder characterized by selective postreceptor insulin resistance.  J Clin Invest. 1998;  101 1111-1120
• 28 Baynes K C, Beeton C A, Panayotou G, Stein R, Soos M, Hansen T, Simpson H, O’Rahilly S, Shepherd P R, Whitehead J P. Natural variants of human p85 alpha phosphoinositide 3-kinase in severe insulin resistance: a novel variant with impaired insulin-stimulated lipid kinase activity.  Diabetologia. 2000;  43 321-331
• 29 Kausch C, Hamann A, Uphues I, Niendorf A, Muller-Wieland D, Joost H G, Algenstaedt P, Dreyer M, Rudiger H W, Haring H U, Eckel J, Matthaei S. Association of impaired phosphatidylinositol 3-kinase activity in GLUT1- containing vesicles with malinsertion of glucose transporters into the plasma membrane of fibroblasts from a patient with severe insulin resistance and clinical features of Werner syndrome.  J Clin Endocrinol Metab. 2000;  85 905-918
• 30 Hoepffner H J, Dreyer M, Reimers U, Schmidt-Preuss U, Koepp H P, Rudiger H W. A new familial syndrome with impaired function of three related peptide growth factors.  Hum Genet. 1989;  83 209-216
• 31 Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.  Nature. 1970;  227 680-685
• 32 Katagiri H, Asano T, Ishihara H, Inukai K, Shibasaki Y, Kikuchi M, Yazaki Y, Oka Y. Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3- kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes.  J Biol Chem. 1996;  271 16 987-16 990
• 33 Mendez R, Myers M G Jr, White M F, Rhoads R E. Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase.  Mol Cell Biol. 1996;  16 2857-2864
• 34 Cheatham B, Vlahos C J, Cheatham L, Wang L, Blenis J, Kahn C R. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.  Mol Cell Biol. 1994;  14 4902-4911
• 35 Bjornholm M, Kawano Y, Lehtihet M, Zierath J R. Insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after in vivo insulin stimulation.  Diabetes. 1997;  46 524-527
• 36 Goodyear L J, Giorgino F, Sherman L A, Carey J, Smith R J, Dohm G L. Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects.  J Clin Invest. 1995;  95 2195-2204
• 37 Rondinone C M, Wang L M, Lonnroth P, Wesslau C, Pierce J H, Smith U. Insulin receptor substrate (IRS) 1 is reduced and IRS-2 is the main docking protein for phosphatidylinositol 3-kinase in adipocytes from subjects with non-insulin-dependent diabetes mellitus.  Proc Natl Acad Sci USA. 1997;  94 4171-4175
• 38 Hansen L, Echwald S M, Hansen T, Urhammer S A, Clausen J O, Pedersen O. Amino acid polymorphisms in the ATP-regulatable inward rectifier Kir6.2 and their relationships to glucose- and tolbutamide-induced insulin secretion, the insulin sensitivity index, and NIDDM.  Diabetes. 1997;  46 508-512
• 39 Hansen T, Andersen C B, Echwald S M, Urhammer S A, Clausen J O, Vestergaard H, Owens D, Hansen L, Pedersen O. Identification of a common amino acid polymorphism in the p85alpha regulatory subunit of phosphatidylinositol 3-kinase: effects on glucose disappearance constant, glucose effectiveness, and the insulin sensitivity index.  Diabetes. 1997;  46 494-501
• 40 Baier L J, Wiedrich C, Hanson R L, Bogardus C. Variant in the regulatory subunit of phosphatidylinositol 3-kinase (p85alpha): preliminary evidence indicates a potential role of this variant in the acute insulin response and type 2 diabetes in Pima women.  Diabetes. 1998;  47 973-975
• 41 Almind K, Delahaye L, Hansen T, van Obberghen E, Pedersen O, Kahn C R. Characterization of the Met³²⁶Ile variant of phosphatidylinositol 3- kinase p85alpha.  Proc Natl Acad Sci USA. 2002;  99 2124-2128