Binding , Internalization , and Lysosomal Association of 125 I-Glucagon in Isolated Rat Hepatocytes

INTRODUCTION Glucagon is a polypeptide hormone secreted by the A cell of the islets of Langerhans and gastrointestinal This work was performed while Dr. P. Gorden was visiting professor at the Institute of Histology and Embryology, University of Geneva, Switzerland. Receivedfor publication 17 December 1979 and in revised form 14 July 1980. tract that plays an important role in fuel homeostasis. The hormone produces these metabolic effects by activating the enzyme adenylate cyclase to form cyclic 3'5'-AMP, which in turn activates a series of phosphorylation steps (1-3). The essential components of this system, i.e., the receptor, the catalytic component, and other regulatory components, are contained in the plasma membrane; and glucagon is a classic example of a polypeptide hormone that activates a second messenger (3). It has been demonstrated recently by quantitative electron microscope (EM)l autoradiography in rat hepatocytes in vitro and in rat liver in vivo that 125Iinsulin binds to the plasma membrane of the hepatocyte and is subsequently internalized by the cell (4-8). Insulin, in contrast to glucagon, has no known second messenger and the mechanism involved in its activation of intracellular processes is unknown. In this investigation we have studied the morphologic events involved in the interaction of 125I-glucagon with freshly isolated rat hepatocytes and have compared these events with insulin and other polypeptide hormones. METHODS Cells and reagents. Hepatocytes were isolated from 6-8wk-old male Wistar rats fed ad lib. by a modification of the method of Seglen (9). The cells were suspended in buffer, then washed four times by low-speed centrifugation. 95% of the cell suspension obtained by this procedure are hepatocytes and 85% of these hepatocytes are viable as judged by two criteria: their ability to exclude trypan blue I Abbreviation used in this paper: EM, electron microscope. 1081 J. Clin. Invest. ( The American Society for Clinical Investigation, Inc. * 0021-9738/80/11/1081/13 $1.00 Volume 66 November 198