Insulin Binding and Metabolism by Hepatocytes in Primary Culture

INSULIN BINDING AND METABOLISM BY HEPATOCYTES IN PRIMARY CULTURE

N. Kalant

TABLE OF CONTENTS

I. Introduction 182

II. The Nature of Binding of Insulin to Cultured Hepatocytes 182 A. Binding 182 B. Dissociation 183 C. Relation of Binding to Stimulation of Glycogenesis 185

III. Degradation of Insulin 185 A. Degradation of Insulin by Lysosomal Enzymes 187 B. Fate of Intralysosomal Insulin 187 C. Susceptibility of Receptor-Bound Insulin to Cytosolic Insulin Degrading

Enzymes 188 D. Effect of Chloroquine on Insulin Degradation 188

IV. Effects of Exposure to Insulin 191 A. Down-Regulation of Binding 192 B. Effect on Insulin Degradation 193 C. Effect on Insulin-Stimulated Glycogenesis 195

V. Discussion and Conclusions 197

Acknowledgments 198

References 198

I. INTRODUCTION

In the past decade there has been an increasing use of primary cell cultures to study biochemical and biological characteristics of hepatocytes. This cell preparation offers a number of advantages over freshly isolated hepatocytes and long-term cultures: freshly isolated hepatocytes suffer variable damage to their plasma membrane during the process of isolation, leading to a decrease in hormone binding' and to leakage of enzymes into the incubation medium;' culture for a short period permits repair of this damage. Furthermore because these cells survive for a number of days in culture, it is possible to carry out certain types of study which are not feasible in short-term incubations of freshly isolated hepatocytes. On the other hand, cells in primary culture retain normal hepatocyte morphology and major liver-specific functions such as albumin synthesis, gluconeogenesis, urea cycle activity, and hormone-specific responses, whereas many of these characteristics are lost in established, replicating cell lines, derived usually from hepatomas.