An approach involving micro'ijection and microanalysis has been developed to investigate signaltransduction pathways involved in the hormonal control of metabolism. We have applied this strategy to investigate the role ofRas signaling in the acute activation of glucose transport by insulin in cardiac myocytes. Glucose transport activity was assessed by measuring the initial rate of accumulation of 2-deoxyglucose 6-phosphate (dGlc6P) in individual cells after incubation in 2-deoxyglucose. Insulin increased accumulation of dGlc6P by 3-to 4-fold, consistent with its stimulatory effect on glucose transport. Accumulation of dGlc6P was increased severalfold by microin'ecting the nonhydrolyzable GTP analogue, guanosine 5'-[ythioltriphosphate, which activates members of the Ras superfamily of GTP-binding proteins. Injecting activated Ha-Ras protein also mimicked insulin by increasing dGlc6P; whereas, iu'ecting a Ras protein lacking the COOH-terminal site of fatty acylation required for Ras function was without effect. Introducing the neutralizing Ras antibody Y13-259 into cells attenuated the effect of insulin. These findings implicate Ras in the acute regulation of metabolism by insulin.Blood glucose levels are maintained within a relatively narrow range through the actions of several hormones, of which insulin is the most important (1). A rise in blood sugar promotes release of insulin, which stimulates glucose transport in skeletal muscle fibers, cardiac myocytes, and adipocytes (2, 3). Insulin increases glucose transport by causing translocation of glucose transporter isoform 4 (GLUT4) from intracellular compartments to the plasma membrane (4, 5). The effect on GLUT4 translocation reaches a maximum within minutes of an increase in the insulin concentration. Several other processes are rapidly changed in response to the hormone (6, 7). For example, glycogen synthesis is increased in skeletal muscle and fat cells, lipogenesis is increased in adipocytes and hepatocytes, and gluconeogenesis is decreased in hepatocytes. These acute metabolic actions of insulin are essential for the efficient uptake and storage of excess blood glucose.Insulin elicits other actions that develop with time courses of many minutes to hours (6). Such effects are observed in a wide variety of cell types and are associated with increased growth and/or differentiation of cells. There is increased evidence that these actions of insulin involve Ras, a GTP-binding protein that is a transducer of diverse physiological signals (8, 9). Insulin increases the proportion of Ras in the active GTP-bound state (10-12) via a mechanism that appears to involve formation of a complex between insulin receptor substrate 1 (IRS-1), guanine nucleotide-releasing factor mSOS, and growth factor receptor-bound protein (GRB2) (13)(14)(15)(16) (22). However, the potential of microinjection in investigating the important metabolic actions of insulin has not been exploited because of problems associated with measuring responses in the cells that have been injected. ...