Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes.

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease

HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings

Molecular analysis of insulin action using high throughput genetic screens

The insulin-stimulated uptake of glucose by muscle and adipose is vital for the maintenance of glucosehomeostasis in the body. This uptake primarily occurs through the action of the insulin-regulatable glucosetransporter GLUT4, which is rapidly translocated to the plasma membrane in response to the insulin signal.However, in the insulin resistant state, insulin is unable to effect a normal biological response in its targettissues, characterized by decreased glucose uptake as a result of defects in insulin signaling and attenuatedGLUT4 translocation. Elucidating the molecular causes underlying insulin resistance is important for thedevelopment of therapeutics for this disease.Identifying the components involved in the propagation or regulation of insulin signaling is an important step inunderstanding insulin resistance. To date, the upstream components of the signaling pathway are wellestablished, demonstrating the importance of the insulin receptor, IRS proteins and PI3K/Akt signaling axis inthis process. As a result much work has focused on defects at the point of IRS in the development of insulinresistance. However, it has recently been suggested that defects associated with insulin resistance occurindependently of IRS. Therefore, identifying the sites that this dysfunction occurs at is of great interest inunderstanding this disease.The purpose of this study is to discover novel proteins involved in the regulation of the insulin signaling andGLUT4 translocation. A GLUT4-overexpressing HeLa cell line was developed and optimized for use in highthroughput screening for regulators of insulin stimulated GLUT4 translocation. Insulin stimulation causedGLUT4 translocation to the plasma membrane, and the activation of the PI3K/Akt signaling pathway in thesecells. Using this cell line, I performed an siRNA screen of kinase and DUB libraries which identified a number ofnovel targets that may play a role in the regulation of insulin stimulated GLUT4 translocation. In conclusion,this assay can be used to identify novel regulators of insulin stimulated GLUT4 translocation, which maypotentially represent targets for drug development in the treatment of insulin resistanc

Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes.

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease

Ministry of Health clinical practice guidelines: Depression

Singapore Medical Journal532137-144SIMJ

Genetic risk of extranodal natural killer T-cell lymphoma: a genome-wide association study

10.1016/S1470-2045(16)30148-6LANCET ONCOLOGY1791240-1247United State