The Serine/Threonine protein-kinase Akt plays a central role in the modulation of glucose metabolism, apoptosis and cell proliferation. In the heart, two highly conserved isoforms, Akt1 and Akt2, are expressed. These may exert different functions under physiological conditions as well as during formation of adaptive/maladaptive cardiac hypertrophy. The third isoform, Akt3, is not detectable in heart and HL-1 cells. The aim of the study is to investigate the isoform-specific functions of Akt1 and Akt2.

With lentiviral vectors, which contain shRNA against Akt1 (DAkt1) and Akt2 (DAkt2), we stably transfected HL-1 cells, a murine cardiomyocyte cell line. After selection with puromycin, the verification of the knock down and the expression analysis were done by western blotting using the infrared imaging system Odyssey (LI-COR Biosciences). A cell line transfected with non-target shRNA (shc) was used as a control. The successful knock down of the Akt-isoforms resulted in a reduction of the protein level in the range of 85–90% for either isoform. The knock down of one isoform did not influence the expression of the other isoform or upregulate Akt3. Quantification of whole Akt content in the different cell lines revealed that Akt1 represents 80% of total Akt in shc cells. Akt2 only accounts for 20%. Analysis of the phosphorylation levels of Serin473 reveals that DAKT1 and untransfected HL-1 show equal phosphorylation levels, 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. Thus, insulin or IGF-induced stimulation results principally in phosphorylation of Akt2. To investigate downstream targets of Akt signalling we examined the phosphorylation of the direct Akt-target GSK3ß. The stimulation with insulin or IGF led to an enhanced phosphorylation of GSK3ß at Serin9. The knock down of either Akt isoform resulted in a similar reduction of GSK3ß phosphorylation, though the expression was not influenced. In response to insulin/IGF, Akt2 has been linked to specifically modulate glucose uptake. We therefore investigated to what degree Akt regulates the expression of insulin-responsive glucose transporter GLUT4 and the Rab-GTPase AS160, which modulates the translocation of GLUT4 to the sarcolemma.

We could show that the expression of GLUT4 protein was significantly reduced in both knock down lines in comparison to shc, but this was more pronounced in DAkt2 cells. AS160 was significantly suppressed in DAkt2 cells compared to shc whereas its expression remained unchanged in DAkt1 cells. Our data show, that although Akt1 represents the highest expressed Akt-isoform, Akt2 is phosphorylated predominantly by insulin or IGF. Thus, on the functional level Akt2 appears to play a major role, especially in the context of glucose metabolism.