To elucidate the functions of the serine/threonine kinase Akt/PKB in vivo, we generated mice lacking both akt1 and akt2 genes. Akt1/Akt2 double-knockout (DKO) mice exhibit severe growth deficiency and die shortly after birth. These mice display impaired skin development because of a proliferation defect, severe skeletal muscle atrophy because of a marked decrease in individual muscle cell size, and impaired bone development. These defects are strikingly similar to the phenotypes of IGF-1 receptor-deficient mice and suggest that Akt may serve as the most critical downstream effector of the IGF-1 receptor during development. In addition, Akt1/Akt2 DKO mice display impeded adipogenesis. Specifically, Akt1 and Akt2 are required for the induced expression of PPAR␥, the master regulator of adipogenesis, establishing a new essential role for Akt in adipocyte differentiation. Overall, the combined deletion of Akt1 and Akt2 establishes in vivo roles for Akt in cell proliferation, growth, and differentiation. These functions of Akt were uncovered despite the observed lower level of Akt activity mediated by Akt3 in Akt1/Akt2 DKO cells, suggesting that a critical threshold level of Akt activity is required to maintain normal cell proliferation, growth, and differentiation.

The serine/threonine kinase Akt is an upstream positive regulator of the mammalian target of rapamycin (mTOR). However, the mechanism by which Akt activates mTOR is not fully understood. The known pathway by which Akt activates mTOR is via direct phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2), which is a negative regulator of mTOR. Here we establish an additional pathway by which Akt inhibits TSC2 and activates mTOR. We provide for the first time genetic evidence that Akt regulates intracellular ATP level and demonstrate that Akt is a negative regulator of the AMP-activated protein kinase (AMPK), which is an activator of TSC2. We show that in Akt1/Akt2 DKO cells AMP/ATP ratio is markedly elevated with concomitant increase in AMPK activity, whereas in cells expressing activated Akt there is a dramatic decrease in AMP/ATP ratio and a decline in AMPK activity. Currently, the Akt-mediated phosphorylation of TSC2 and the inhibition of AMPK-mediated phosphorylation of TSC2 are viewed as two separate pathways, which activate mTOR. Our results demonstrate that Akt lies upstream of these two pathways and induces full inhibition of TSC2 and activation of mTOR both through direct phosphorylation and by inhibition of AMPK-mediated phosphorylation of TSC2. We propose that the activation of mTOR by Akt-mediated cellular energy and inhibition of AMPK is the predominant pathway by which Akt activates mTOR in vivo.The serine/threonine protein kinase Akt, also known as protein kinase B, a downstream effector of phosphoinositide-3-OH kinase, has emerged as a critical mediator of the mammalian target of rapamycin (mTOR) 2 activity. Mammalian cells express three separate Akt proteins (Akt1-3), which share Ͼ80% amino acid sequence identity and are encoded by different genes. The rate-limiting step in Akt activation is the binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of Akt and the subsequent translocation of Akt to the plasma membrane. Akt is then phosphorylated by 3-phosphoinositide-dependent kinase-1 and by another as yet unknown phosphoinositide-3-OH kinase-dependent kinase. Both phosphorylation events are required for full activation of Akt (for reviews see Refs. 1-3). Biochemical and genetic data show that Akt is a positive regulator of mTOR that mediates the activation of mTOR by growth factors (reviewed in Ref. 4). mTOR controls mRNA translation by phosphorylating and activating S6 kinase 1 (S6K1) and by phosphorylating and inactivating the eukaryotic initiation factor 4E-binding proteins (4E-BPs), which repress mRNA translation. Thus, the phosphorylation status of S6K1 and one of the 4E-BPs members, 4E-BP1, is often used as readout for mTOR activity in vivo. mTOR is activated by the small GTPase Rheb, which is inhibited by its GAP protein TSC2 that heterodimerizes with tuberous sclerosis complex 1 (TSC1) (5-7). Genetic studies and biochemical analyses in mammalian cells (8 -12) and Drosophila (11, 13), show that TSC2 is an upstream negative regulator of mTOR. Akt...

Akt contributes to tumorigenesis by inhibiting apoptosis. Here we establish that Akt is required for normal cell proliferation and susceptibility to oncogenesis independently of its antiapoptotic activity. Partial ablation of Akt activity by deleting Akt1 inhibits cell proliferation and oncogenesis. These effects are compounded by deleting both Akt1 and Akt2. In vivo, Akt1 null mice are resistant to MMTV-v-H-Ras-induced tumors and to skin carcinogenesis. Thus, partial ablation of Akt activity is sufficient to suppress tumorigenesis in vitro and in vivo. The effect of Akt deficiency on cell proliferation and oncogenesis is p53 independent but mTORC1 dependent. Surprisingly, upon mTORC1 hyperactivation, the reduction in Akt activity does not impair cell proliferation and susceptibility to oncogenic transformation; thus, Akt may mediate these processes exclusively via mTORC1.

Background Delayed gadolinium (Gd) enhancement MRI (DEMRI) identifies non-viable myocardium, but is non-specific and may overestimate nonviable territory. Manganese (Mn2+)-enhanced MRI (MEMRI) denotes specific Mn2+ uptake into viable cardiomyocytes. We performed a dual-contrast myocardial assessment in a porcine ischemia-reperfusion (IR) model to test the hypothesis that combined DEMRI and MEMRI will identify viable infarct border zone (BZ) myocardium in vivo. Methods and Results Sixty-minute LAD ischemia-reperfusion injury (IR) was induced in 13 adult swine. Twenty-one days post-IR, 3T cardiac MRI was performed. MEMRI was obtained after injection (0.7 cc/kg) of Mn2+ contrast agent (EVP1001-1, Eagle Vision Pharmaceutical Corp.). DEMRI was then acquired after 0.2mmol/kg Gd injection. Left ventricular (LV) mass, infarct, and function were analyzed. Subtraction of MEMRI defect from DEMRI signal identified injured border zone myocardium. Explanted hearts were analyzed by 2,3,5-triphenyltetrazolium chloride (TTC) stain and tissue electron microscopy (TEM) to compare infarct, BZ, and remote myocardium. Average LV ejection fraction was reduced (30±7%). MEMRI and DEMRI infarct volumes correlated with TTC (MEMRI: r=0.78; DEMRI: r=0.75; p<0.004). MEMRI infarct volume percentage was significantly lower than DEMRI (14±4%* vs. 23±4%; *p<0.05). BZ MEMRI SNR was intermediate to remote and core infarct SNR (7.5±2.8* vs. 13.2±3.4 and 2.9±1.6; *p<0.0001), and DEMRI BZ SNR tended to be intermediate to remote and core infarct (8.4±5.4 vs. 3.3±0.6 and 14.3±6.6; p>0.05). TEM analysis exhibited preserved cell structure in BZ cardiomyocytes despite transmural DEMRI enhancement. Conclusions Dual-contrast MEMRI-DEMRI detects BZ viability within DEMRI infarct zones. This approach may identify injured, at-risk myocardium in ischemic cardiomyopathy.