Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function.

A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34(f/f) mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34(f/f) mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34(f/f) mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.

Ablation of PI3K p110-α prevents high-fat diet-induced liver steatosis.

OBJECTIVE: To determine whether the phosphoinositide 3-kinase (PI3K) catalytic subunits p110-α and p110-ß play a role in liver steatosis induced by a high-fat diet (HFD). RESEARCH DESIGN AND METHODS: Liver-specific p110-α and p110-ß knockout mice and control animals for each group were fed an HFD or normal chow for 8 weeks. Biochemical assays and quantitative real-time PCR were used to measure triglyceride, expression of lipogenic and gluconeogenic genes, and activity of protein kinases downstream of PI3K in liver lysates. Fatty acid uptake and incorporation into triglycerides were assessed in isolated hepatocytes. RESULTS: Hepatic triglyceride levels in HFD-fed p110-α(-/-) mice were 84 ± 3% lower than in p110-α(+/+) mice, whereas the loss of p110-ß did not significantly alter liver lipid accumulation. p110-α(-/-) livers also showed a reduction in atypical protein kinase C activity and decreased mRNA and protein expression of several lipogenic genes. Hepatocytes isolated from p110-α(-/-) mice exhibited decreased palmitate uptake and reduced fatty acid incorporation into triglycerides as compared with p110-α(+/+) cells, and hepatic expression of liver fatty acid binding protein was lower in p110-α(-/-) mice fed the HFD as compared with controls. Ablation of neither p110-α nor p110-ß ameliorated glucose intolerance induced by the HFD, and genes involved in gluconeogenesis were upregulated in the liver of both knockout animals. CONCLUSIONS: PI3K p110-α, and not p110-ß, promotes liver steatosis in mice fed an HFD. p110-α might exert this effect in part through activation of atypical protein kinase C, upregulation of lipogenesis, and increased uptake of fatty acids.

Inhibition of mammalian target of rapamycin signaling by 2-(morpholin-1-yl)pyrimido[2,1-alpha]isoquinolin-4-one.

Signaling through the mammalian target of rapamycin (mTOR) is hyperactivated in many human tumors, including hamartomas associated with tuberous sclerosis complex (TSC). Several small molecules such as LY294002 inhibit mTOR kinase activity, but they also inhibit phosphatidylinositol 3-kinase (PI3K) at similar concentrations. Compound 401 is a synthetic inhibitor of DNA-dependent protein kinase (DNA-PK) that also targets mTOR but not PI3K in vitro (Griffin, R. J., Fontana, G., Golding, B. T., Guiard, S., Hardcastle, I. R., Leahy, J. J., Martin, N., Richardson, C., Rigoreau, L., Stockley, M., and Smith, G. C. (2005) J. Med. Chem. 48, 569-585). We used 401 to test the cellular effect of mTOR inhibition without the complicating side effects on PI3K. Treatment of cells with 401 blocked the phosphorylation of sites modified by mTOR-Raptor and mTOR-Rictor complexes (ribosomal protein S6 kinase 1 Thr(389) and Akt Ser(473), respectively). By contrast, there was no direct inhibition of Akt Thr(308) phosphorylation, which is dependent on PI3K. Similar effects were also observed in cells that lack DNA-PK. The proliferation of TSC1-/- fibroblasts was inhibited in the presence of 401, but TSC1+/+ cells were resistant. In contrast to rapamycin, long-term treatment of TSC1-/- cells with 401 did not up-regulate phospho-Akt Ser(473). Because increased Akt activity promotes survival, this may explain why the level of apoptosis was increased in the presence of 401 but not rapamycin. These results suggest that mTOR kinase inhibitors might be more effective than rapamycins in controlling the growth of TSC hamartomas and other tumors that depend on elevated mTOR activity.

Apoptotic proteins in the temporal cortex in schizophrenia: high Bax/Bcl-2 ratio without caspase-3 activation.

OBJECTIVE: Neuroimaging findings have identified lower cortical gray matter volume in schizophrenia. Apoptosis (programmed cell death) has been proposed as a contributing pathophysiological mechanism. Levels of antiapoptotic Bcl-2 protein are low in the temporal cortex of schizophrenia patients. Bcl-2 interacts with the proapoptotic Bax protein at an upstream checkpoint to regulate the activation of apoptosis by caspase-3 and other proteolytic caspase proteins. A high Bax/Bcl-2 ratio is associated with greater vulnerability to apoptotic activation, while a high caspase-3 level is often associated with apoptotic activity. It was hypothesized that the Bax/Bcl-2 ratio, but not caspase-3, would be high in the temporal cortex of patients with chronic schizophrenia. METHOD: Bax, Bcl-2, and caspase-3 proteins were measured by semiquantitative Western blot in Brodmann's area 21 (middle temporal gyrus) of postmortem tissue from patients with schizophrenia (N=15), bipolar disorder (N=15), or major depression (N=15) and nonpsychiatric comparison subjects (N=15). RESULTS: The Bax/Bcl-2 ratio was 50% higher in the schizophrenia patients than the nonpsychiatric comparison subjects. The level of caspase-3 (inactive zymogen and activated subunits) was not significantly different. CONCLUSIONS: The higher Bax/Bcl-2 ratio suggests that cortical cells are vulnerable to apoptosis in chronic schizophrenia. However, the normal caspase-3 level suggests that apoptosis is not active in this illness phase. Furthermore, the results appear to distinguish the pathophysiology of schizophrenia from most classic neurodegenerative disorders, in which postmortem caspase-3 levels are high. Further studies are needed to investigate the implications of abnormal apoptotic proteins in schizophrenia.