DIABETES, VOL. 48, FEBRUARY 1999 403 Mutational Analysis of the Coding Regions of the Genes Encoding Protein Kinase B- and - , Phosphoinositide-Dependent Protein Kinase-1, Phosphatase Targeting to Glycogen, Protein Phosphatase Inhibitor-1, and Glycogenin Lessons Fro m a Search fo r Genetic Variability o f the Insulin-Stimulated Glycogen Synthesis Pathway of Skeletal Muscle in NIDDM Patients Lars Hansen, Helle Fjordvang, Søren K. Rasmussen, Henrik Vestergaard, Søren M. Echwald, Torben Hansen, Dario Alessi, Shirish Shenolikar, Alan R. Saltiel, Fabrizio Barbetti, and Oluf Pedersen The finding of a reduced insulin-stimulated glucose uptake and glycogen synthesis in the skeletal muscle of glucose-tolerant first-degree relatives of patients with NIDDM, as well as in cultured fibroblasts and skeletal muscle cells isolated from NIDDM patients, has been interpreted as evidence for a genetic involvement in the disease. The mode of inheritance of the common forms of NIDDM is as yet unclear, but the prevailing hypothesis supports a polygenic model. In the present st udy, we tested the hypothesis that the putative inher- itable defects of insulin-stimulated muscle glycogen synthesis might be caused by genetic variability in the genes encoding proteins shown by biochemical evidence to be involved in insulin-stimulated glycogen synthesis in skeletal muscle. In 70 insulin-resistant Danish NIDDM patients, mutational analysis by reverse tran- scription–polymerase chain reaction–single strand con- formation polymorphism–heteroduplex analysis was performed on genomic DNA or skeletal muscle–derived cDNAs encoding glycogenin, protein phosphatase inhibit or-1, phophatase targeting to glycogen, protein kinase B- and - , and the phosphoinositide-dependent protein kinase-1. Although a number of silent variants were identified in some of the examined genes, we found no evidence for the hypothesis that the defective insulin-stimulated glycogen synthesis in skeletal muscle in NIDDM is caused by structural changes in the genes encoding the known components of the insulin-sensitive glycogen synthesis pathway of skeletal muscle. Dia - betes 48:403–407, 1999 S trong evidence for a genetic involvement in the pathogenesis of NIDDM comes from the studies of first-degree relatives of Caucasian NIDDM patients. These glucose-tolerant first-degree rela- tives show a significantly reduced sensitivity to insulin when examined by both the hyperinsulinemic-euglycemic clamp and the intravenous glucose tolerance test (1,2). Prospec- tive studies suggest that this reduction in insulin sensitivity precedes the development of NIDDM in Caucasians (2). Hyperinsulinemic-euglycemic or hyperinsulinemic-hyper- glycemic clamp studies in combination with either indirect calorimetry (1,3) o r nuclear magnetic resonance show that the insulin resistance of peripheral tissue (i.e., primarily muscle tissue) is caused both by a reduction in insulin-stimulated glu- cose uptake and phosphorylation and by a reduction in glycogen synthesis (4,5). Biochemically, this decrease in insulin-stimulated glycogen synthesis has been associated with a defective in vivo activation of muscle glycogen synthase in response to insulin (2,3,6). Similarly, studies of cultured muscle cells from NIDDM patients and matched control sub- jects have shown impaired insulin-stimulated glycogen syn- thesis in NIDDM (7,8). These findings suggest that in NIDDM, defective signaling mechanisms may result from decreased From the Steno Diabetes Center and Hagedorn Research Institute (L.H., H.F., S.K.R., H.V., S.M.E., T.H., O.P.), Gentofte, Denmark; the Department of Biochemistry (D.A.), Medical Sciences Institute, The University of Dundee, Scotland, U.K.; the Department of Pharmacology and Cancer Biology (S.S.), Duke University Medical Center, Durham, North Carolina; the Department o f Physiology (A.R.S.), University of Michigan Scho ol o f Med- icine, Ann Arbor, Michigan; and the Unit of Molecular Pathology of Diabetes ( F.B.), H. San Raffaele Scientific Institute, Milan, Italy. Address correspondence and reprint requests to Lars Hansen, MD, Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 2-6, DK-2820, Gentofte, Denmark. E-mail: larh@hagedorn.dk. Received for publication 18 May 1998 and accepted in revised form 9 October 1998. Additional information can be found in an on-line appendix at www. d iab e te s .o rg/ d iab e te s / ap p e nd ix .as p . GSK-3, glycogen synthase kinase-3; I-1, protein phosphatase inhibitor-1; IRS-1, insulin receptor substrate-1; PCR, polymerase chain reaction; PDK-1, phosphoinositide-dependent kinase-1; PI 3 K, phosphatidyl inositol 3-kinase; PKB, protein kinase B; PP1, protein phosphatase-1; PP1G M , glycogen-asso- ciated subunit of the type 1 phosphatase; PPP1R5, regulatory subunit of type 1 protein phosphatase; PTG, phosphatase targeting to glycogen; RT, reverse transcription; SSCP, single-strand conformation polymorphism.