The insulin receptor substrate (IRS) proteins are cytoplasmic adaptor molecules that function as signaling intermediates downstream of activated cell surface receptors. Based on data implicating IRS-2 but not IRS-1 in breast cancer invasion, survival, and metastasis, we assessed the contribution of IRS-1 and IRS-2 to aerobic glycolysis, which is known to impact tumor growth and progression. For this purpose, we used tumor cell lines derived from transgenic mice that express the polyoma virus middle T antigen (PyV-MT) in the mammary gland and that are wild-type (WT) or null for either Irs-1 (Irs-1 ؊/؊ ) or Irs-2 (Irs-2 ؊/؊ ). Aerobic glycolysis, as assessed by the rate of lactic acid production and glucose consumption, was diminished significantly in Irs-2 ؊/؊ cells when compared with WT and Irs-1 ؊/؊ cells. Expression of exogenous Irs-2 in Irs-2 ؊/؊ cells restored the rate of glycolysis to that observed in WT cells. The transcription factor FoxO1 does not appear to be involved in Irs-2-mediated glycolysis. However, Irs-2 does regulate the surface expression of glucose transporter 1 (Glut1) as assessed by flow cytometry using a Glut1-specific ligand. Suppression of Glut1 expression inhibits Irs-2-dependent invasion, which links glycolysis to mammary tumor progression. Irs-2 was shown to be important for mammalian target of rapamycin (mTor) activation, and Irs-2-dependent regulation of Glut1 surface expression is rapamycin-sensitive. Collectively, our data indicate that Irs-2, but not Irs-1, promotes invasion by sustaining the aerobic glycolysis of mouse mammary tumor cells and that it does so by regulating the mTor-dependent surface expression of Glut1.Glucose metabolism in cancer cells differs significantly from that of normal cells as observed initially by Warburg in the 1920s (1). Specifically, cancer cells depend more on glycolysis than oxidative phosphorylation to generate ATP, even in high oxygen tensions. Subsequent studies have affirmed the importance of aerobic glycolysis in tumor progression and have shown that it provides tumor cells with a selective advantage in their ability to progress toward invasive and metastatic disease (2-4). Significantly, the "Warburg effect" has become a powerful and standard imaging technique for detecting tumors and their metastases by positron emission tomography using [ 18 F]deoxyglucose (5). Aerobic glycolysis is also an ideal target for therapeutic intervention because apoptosis results if this process is perturbed (2, 6). To exploit aerobic glycolysis for the clinical management of cancer, however, much more needs to be learned about how this form of metabolism is induced and sustained in tumors. Our interest is in understanding how aerobic glycolysis is regulated in breast cancer.The insulin receptor substrate (IRS) 3 proteins are cytoplasmic adaptor molecules that function as signaling intermediates downstream of activated cell surface receptors (7). The role of the IRS-2 family member is of particular significance in breast cancer. IRS-2 is predominantly expressed ...