Production of Inhibitor of Insulin-Receptor Tyrosine Kinase in Fibroblasts From Patient With Insulin Resistance and NIDDM PAOLO SBRACCIA, PATRICIA A. GOODMAN, BETTY A. MADDUX, K.Y. WONG, Y.-D.l. CHEN, GERALD M. REAVEN, AND IRA D. GOLDFINE Although non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in insulin action, the molecular basis of this resistance is unknown. We studied fibroblasts from a markedly insulin-resistant patient with NIDDM but without acanthosis nigricans. Her fibroblasts were resistant to insulin when a- aminoisobutyric acid uptake was measured. Fibroblasts from this patient demonstrated normal insulin-receptor content as measured by both insulin- receptor radioimmunoassay and by Scatchard analysis. However, when compared with nondiabetic control subjects, insulin-receptor kinase assays of wheat-germ-purified receptors prepared from her fibroblasts showed very low basal and no insulin- stimulated tyrosine kinase activity. The insulin receptor was then removed from the wheat-germ fraction by monoclonal antibody affinity chromatography. This insulin-receptor-deficient fraction inhibited both basal and insulin-stimulated tyrosine kinase activity of highly purified insulin receptors. When the specificity of this inhibition was tested, less inhibition was seen with insulinlike growth factor l-receptor tyrosine kinase, and even less inhibition was seen with the proto-oncogene p60 csrc tyrosine kinase. Thus, these studies indicate that fibroblasts from an insulin-resistant patient with NIDDM produce a relatively specific glycoprotein inhibitor of insulin-receptor tyrosine kinase. Therefore, these studies raise the possibility that this inhibitor may play an important role in the insulin resistance seen in this patient. Diabetes 40:295-99,1991 From the Division of Diabetes and Endocrine Research, Mount Zion Medical Center of the University of California at San Francisco, and the Division of Gerontology, Department of Medicine, Stanford University School of Medicine, Palo Alto, California. Address correspondence and reprint requests to Ira D. Goldfine, MD, Di- rector, Division of Diabetes and Endocrine Research, Mount Zion Medical Center of the University of California at San Francisco, PO Box 7921, San Francisco, CA 94120. Received for publication 12 October 1990 and accepted in revised form 30 October 1990. N on-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in both insulin secretion and insulin action (1-4). In most patients with NIDDM, the molecular basis of the insulin resist- ance is unknown. The cellular response to insulin is mediated through the insulin receptor (5). The insulin receptor is a tetramer consisting of two identical extracellular a-subunits that bind the hormone and two identical transmembrane (3- subunits that have intracellular tyrosine kinase activity. When insulin binds to the a-subunit, the (3-subunit tyrosine kinase is activated and insulin action ensues (5). A few patients with extreme insulin resistance and acanthosis nigricans have structural abnormalities in their insulin receptors (6- 11), but in most patients with NIDDM, insulin-receptor func- tion appears normal (12,13). In this study, we identified a patient with marked insulin resistance and NIDDM whose fibroblasts produce a factor that inhibits insulin-receptor ki- nase activity. RESEARCH DESIGN AND METHODS Fibroblasts from the patient were obtained by forearm skin biopsy and grown under standard conditions (14). Control fibroblasts from an age- and sex-matched individual were obtained from the National Institute of General Medical Sci- ences Human Genetic Cell Repository (Camden, NJ). Cells were grown in Dulbecco's modified Eagle's medium sup- plemented with 10% fetal bovine serum (Cell Culture Facility, Univ. of California, San Francisco). We purchased A14- 125 l-labeled insulin (2200 Ci/mmol), [7- 32 P]ATP (3000 Ci/mmol), a-[ 3 H]aminoisobutyric acid (AIB; 33 Ci/mmol), and 125 l-labeled Bolton-Hunter reagent from Du Pont-NEN (Chicago, IL); pork insulin from Elanco (Indi- anapolis, IN); and poly(Glu-Tyr) from Sigma (St. Louis, MO). The following were gifts: pure proto-oncogene p60 c ' src from D. Morgan; synthetic insulinlike growth factor I (IGF-I) from Lilly (Indianapolis, IN); and CHO cells transfected with and DIABETES, VOL. 40, FEBRUARY 1991 295 Downloaded from http://diabetesjournals.org/diabetes/article-pdf/40/2/295/358800/40-2-295.pdf by guest on 07 November 2022