Matthew J Brady Introduction

Insulin stimulates the storage of glucose as glycogen in muscle and adipose tissue through the coordinate increase in glucose uptake and modulation of glycogen metabolizing enzymes (1). Insulin binds to its receptor in peripheral tissues and initiates several signaling cascades to increase glucose uptake via translocation of the glucose tranporter-4 (GLUT-4) containing vesicles to the plasma membrane (see Fig. 1). Glucose enters the cells and is phosphorylated by hexokinases to form glucose-6-phosphate (G6P). Depending on the energy requirements of the cell, G6P can be used to generate ATP via glycolysis or be metabolized to uridine diphospho (UDP)-glucose and stored as glycogen. Measurement of glycogen synthesis rates in cells and tissues is thus a powerful way to examine several insulin metabolic responses simultaneously.

Glycogen synthase, the rate-limiting enzyme for glycogen synthesis, catalyzes the incorporation of UDP-glucose into glycogen chains. The enzyme is regulated by both covalent and allosteric mechanisms (2). Glycogen synthase is phosphorylated on multiple residues, resulting in its progressive inactivation. Insulin stimulates glycogen synthase activity by promoting its net dephospho-rylation, through phosphatase activation and kinase inhibition. This action of insulin is facilitated by the stimulation of glucose uptake, because the resulting increase in G6P leads to the allosteric activation of glycogen synthase and increased susceptibility to dephosphorylation (3,4).

3T3-L1 adipocytes are a widely used cell line model for the study of insulin metabolic signaling. These cells are grown as fibroblasts, but they can be ter-

Fig. 1. Dual effect of insulin on glucose metabolism in peripheral tissues. In skeletal muscle and adipose tissues, binding of insulin to its receptor stimulates GLUT-4 vesicle translocation, increasing glucose uptake and intracellular levels of glucose-6-phosphate (G6P). G6P can be used to generate ATP or be metabolized to form uridine diphospho (UDP)-glucose. Insulin also promotes the dephosphorylation and activation of glycogen synthase, which incorporates UDP-glucose into glycogen chains.

Fig. 1. Dual effect of insulin on glucose metabolism in peripheral tissues. In skeletal muscle and adipose tissues, binding of insulin to its receptor stimulates GLUT-4 vesicle translocation, increasing glucose uptake and intracellular levels of glucose-6-phosphate (G6P). G6P can be used to generate ATP or be metabolized to form uridine diphospho (UDP)-glucose. Insulin also promotes the dephosphorylation and activation of glycogen synthase, which incorporates UDP-glucose into glycogen chains.

minally differentiated into lipid-containing adipocytes. Although it may seem counterintuitive to study glycogen metabolism in adipocytes, these cells contain high levels of glucose transporters, insulin receptors, and glycogen synthase and are very metabolically active.

Glucose uptake and storage as glycogen is assayed by adding radioactive glucose as a tracer to the extracellular medium, followed by isolation of cellular glycogen. Glycogen synthase activity is assayed in vitro by measuring the incorporation of UDP-glucose into purified glycogen. These procedures have been adapted for use in 3T3-L1 adipocytes from earlier protocols (5-7).

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