The serine/threonine protein kinase Akt is a key regulator involved in the development of cardiac hypertrophy. To investigate the specific functions of the isoforms Akt1 and Akt2, we generated a set of cell lines derived from HL-1 cardiomyocytes by using lentiviral transfection. These cell lines stably express shRNA directed against Akt1 (DAkt1) and Akt2 (DAkt2). Cells expressing no target shRNA (shC) were used as controls. On the protein level, the successful knock down led to a reduction in the range of 85-90% for either isoform. A compensatory upregulation of the other Akt isoform or Akt3 was not detected. Quantification of whole Akt exhibits, that Akt1 represents 75%, while Akt2 accounts only for 25%. Analysis of the phosphorylation levels of Ser473 reveals that DAkt1 and untransfected HL-1 cells show equal phosphorylation levels after stimulation with insulin or IGF-1 (insulin-like growth factor 1), although only the minor Akt isoform (Akt2) is expressed in DAkt1 cells. On the opposite, in DAkt2 cells a reduced phosphorylation level was observed, though these cells express high levels of Akt1. Phosphorylation of the downstream target GSK3 b(Ser9) was reduced in both knock down cell lines but this effect was not isoform specific. In the context of glucose metabolism, the protein level of insulin regulated GLUT4 glucose transporter was reduced in DAkt1 and DAkt2 cells. In contrast, the protein expression of insulin independent GLUT1 transporter was not influenced in both cell lines. But the Rab GTPase activating protein (GAP) AS160, which regulates the insulin dependent translocation of GLUT4 to the plasma membrane, is specifically reduced on the protein level in DAkt2 cells. Our data show that although Akt1 is the major isoform, Akt2 represents the preferred target activated in HL-1 cells by insulin/IGF-1 stimulation. In HL-1 cardiomyocytes, Akt2 modulates the protein level of AS160 and therefore it appears to play a major role in the context of glucose metabolism.