Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner.

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.

The Caenorhabditis elegans HNF4alpha Homolog, NHR-31, mediates excretory tube growth and function through coordinate regulation of the vacuolar ATPase.

Nuclear receptors of the Hepatocyte Nuclear Factor-4 (HNF4) subtype have been linked to a host of developmental and metabolic functions in animals ranging from worms to humans; however, the full spectrum of physiological activities carried out by this nuclear receptor subfamily is far from established. We have found that the Caenorhabditis elegans nuclear receptor NHR-31, a homolog of mammalian HNF4 receptors, is required for controlling the growth and function of the nematode excretory cell, a multi-branched tubular cell that acts as the C. elegans renal system. Larval specific RNAi knockdown of nhr-31 led to significant structural abnormalities along the length of the excretory cell canal, including numerous regions of uncontrolled growth at sites near to and distant from the cell nucleus. nhr-31 RNAi animals were sensitive to acute challenge with ionic stress, implying that the osmoregulatory function of the excretory cell was also compromised. Gene expression profiling revealed a surprisingly specific role for nhr-31 in the control of multiple genes that encode subunits of the vacuolar ATPase (vATPase). RNAi of these vATPase genes resulted in excretory cell defects similar to those observed in nhr-31 RNAi animals, demonstrating that the influence of nhr-31 on excretory cell growth is mediated, at least in part, through coordinate regulation of the vATPase. Sequence analysis revealed a stunning enrichment of HNF4alpha type binding sites in the promoters of both C. elegans and mouse vATPase genes, arguing that coordinate regulation of the vATPase by HNF4 receptors is likely to be conserved in mammals. Our study establishes a new pathway for regulation of excretory cell growth and reveals a novel role for HNF4-type nuclear receptors in the development and function of a renal system.

Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.

Acoustic liquid handling uses high-frequency acoustic signals that are focused on the surface of a fluid to eject droplets with high accuracy and precision for various life science applications. Here we present a multiwell source plate, the Echo Qualified Reservoir (ER), which can acoustically transfer over 2.5 mL of fluid per well in 25-nL increments using an Echo 525 liquid handler. We demonstrate two Labcyte technologies-Dynamic Fluid Analysis (DFA) methods and a high-voltage (HV) grid-that are required to maintain accurate and precise fluid transfers from the ER at this volume scale. DFA methods were employed to dynamically assess the energy requirements of the fluid and adjust the acoustic ejection parameters to maintain a constant velocity droplet. Furthermore, we demonstrate that the HV grid enhances droplet velocity and coalescence at the destination plate. These technologies enabled 5-µL per destination well transfers to a 384-well plate, with accuracy and precision values better than 4%. Last, we used the ER and Echo 525 liquid handler to perform a quantitative polymerase chain reaction (qPCR) assay to demonstrate an application that benefits from the flexibility and larger volume capabilities of the ER.

Dual manganese-enhanced and delayed gadolinium-enhanced MRI detects myocardial border zone injury in a pig ischemia-reperfusion model.

BACKGROUND: Gadolinium (Gd)-based delayed-enhancement MRI (DEMRI) identifies nonviable myocardium but is nonspecific and may overestimate nonviable territory. Manganese (Mn(2+))-enhanced MRI (MEMRI) denotes specific Mn(2+) 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 identifies viable infarct border zone (BZ) myocardium in vivo. METHODS AND RESULTS: Sixty-minute left anterior descending coronary artery IR injury was induced in 13 adult swine. Twenty-one days post-IR, 3-T cardiac MRI was performed. MEMRI was obtained after injection of 0.7 mL/kg Mn(2+) contrast agent. DEMRI was then acquired after injection of 0.2 mmol/kg Gd. Left ventricular (LV) mass, infarct, and function were analyzed. Subtraction of MEMRI defect from DEMRI signal identified injured BZ myocardium. Explanted hearts were analyzed by 2,3,5-triphenyltetrazolium chloride stain and tissue electron microscopy to compare infarct, BZ, and remote myocardium. Average LV ejection fraction was reduced (30±7%). MEMRI and DEMRI infarct volumes correlated with 2,3,5-triphenyltetrazolium chloride stain analysis (MEMRI, r=0.78; DEMRI, r=0.75; P<0.004). MEMRI infarct volume percentage was significantly lower than that of DEMRI (14±4% versus 23±4%; P<0.05). BZ MEMRI signal-to-noise ratio (SNR) was intermediate to remote and core infarct SNR (7.5±2.8 versus 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 SNR (8.4±5.4 versus 3.3±0.6 and 14.3±6.6; P>0.05). Tissue electron microscopy analysis exhibited preserved cell structure in BZ cardiomyocytes despite transmural DEMRI enhancement. CONCLUSIONS: The dual-contrast MEMRI-DEMRI detects BZ viability within DEMRI infarct zones. This approach may identify injured, at-risk myocardium in ischemic cardiomyopathy.

Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity.

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.

Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2.

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 PPARgamma, 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.