Control of mammalian translation by mRNA structure near caps

The scanning model of RNA translation proposes that highly stable secondary structures within mRNAs can inhibit translation, while structures of lower thermal stability also affect translation if close enough to the 5′ methyl G cap. However, only fragmentary information is available about the dependence of translation efficiency in live mammalian cells on the thermodynamic stability, location, and GC content of RNA structures in the 5′-untranslated region. We devised a two-color fluorescence assay for translation efficiency in single live cells and compared a wide range of hairpins with predicted thermal stabilities ranging from −10 to −50 kcal/mol and 5′ G cap-to-hairpin distances of 1–46 bases. Translation efficiency decreased abruptly as hairpin stabilities increased from ΔG = −25 to −35 kcal/mol. Shifting a hairpin as little as nine bases relative to the 5′ cap could modulate translation more than 50-fold. Increasing GC content diminished translation efficiency when predicted thermal stability and cap-to-hairpin distances were held constant. We additionally found naturally occurring 5′-untranslated regions affected translation differently in live cells compared with translation in in vitro lysates. Our study will assist scientists in designing experiments that deliberately modulate mammalian translation with designed 5′ UTRs

Myosin 5a Is an Insulin-Stimulated Akt2 (Protein Kinase Bβ) Substrate Modulating GLUT4 Vesicle Translocation▿

Phosphatidylinositol 3-kinase activation of Akt signaling is critical to insulin-stimulated glucose transport and GLUT4 translocation. However, the downstream signaling events following Akt activation which mediate glucose transport stimulation remain relatively unknown. Here we identify an Akt consensus phosphorylation motif in the actin-based motor protein myosin 5a and show that insulin stimulation leads to phosphorylation of myosin 5a at serine 1650. This Akt-mediated phosphorylation event enhances the ability of myosin 5a to interact with the actin cytoskeleton. Small interfering RNA-induced inhibition of myosin 5a and expression of dominant-negative myosin 5a attenuate insulin-stimulated glucose transport and GLUT4 translocation. Furthermore, knockdown of Akt2 or expression of dominant-negative Akt (DN-Akt) abolished insulin-stimulated phosphorylation of myosin 5a, inhibited myosin 5a binding to actin, and blocked insulin-stimulated glucose transport. Taken together, these data indicate that myosin 5a is a newly identified direct substrate of Akt2 and, upon insulin stimulation, phosphorylated myosin 5a facilitates anterograde movement of GLUT4 vesicles along actin to the cell surface

GRK2 is an endogenous protein inhibitor of the insulin signaling pathway for glucose transport stimulation

G protein-coupled receptor kinases (GRKs) represent a class of proteins that classically phosphorylate agonist-activated G protein-coupled receptors, leading to uncoupling of the receptor from further G protein activation. Recently, we have reported that the heterotrimeric G protein α-subunit, Gαq/11, can mediate insulin-stimulated glucose transport. GRK2 contains a regulator of G protein signaling (RGS) domain with specificity for Gαq/11. Therefore, we postulated that GRK2 could be an inhibitor of the insulin signaling cascade leading to glucose transport in 3T3-L1 adipocytes. In this study, we demonstrate that microinjection of anti-GRK2 antibody or siRNA against GRK2 increased insulin-stimulated insulin-responsive glucose transporter 4 (GLUT4) translocation, while adenovirus-mediated overexpression of wild-type or kinase-deficient GRK2 inhibited insulin-stimulated GLUT4 translocation as well as 2-deoxyglucose uptake. Importantly, a mutant GRK2 lacking the RGS domain was without effect. Taken together, these results indicate that through its RGS domain endogenous GRK2 functions as a negative regulator of insulin-stimulated glucose transport by interfering with Gαq/11 signaling to GLUT4 translocation. Furthermore, inhibitors of GRK2 can lead to enhanced insulin sensitivity

SIRT1 Exerts Anti-Inflammatory Effects and Improves Insulin Sensitivity in Adipocytes▿ †

SIRT1 is a prominent member of a family of NAD+-dependent enzymes and affects a variety of cellular functions ranging from gene silencing, regulation of the cell cycle and apoptosis, to energy homeostasis. In mature adipocytes, SIRT1 triggers lipolysis and loss of fat content. However, the potential effects of SIRT1 on insulin signaling pathways are poorly understood. To assess this, we used RNA interference to knock down SIRT1 in 3T3-L1 adipocytes. SIRT1 depletion inhibited insulin-stimulated glucose uptake and GLUT4 translocation. This was accompanied by increased phosphorylation of JNK and serine phosphorylation of insulin receptor substrate 1 (IRS-1), along with inhibition of insulin signaling steps, such as tyrosine phosphorylation of IRS-1, and phosphorylation of Akt and ERK. In contrast, treatment of cells with specific small molecule SIRT1 activators led to an increase in glucose uptake and insulin signaling as well as a decrease in serine phosphorylation of IRS-1. Moreover, gene expression profiles showed that SIRT1 expression was inversely related to inflammatory gene expression. Finally, we show that treatment of 3T3-L1 adipocytes with a SIRT1 activator attenuated tumor necrosis factor alpha-induced insulin resistance. Taken together, these data indicate that SIRT1 is a positive regulator of insulin signaling at least partially through the anti-inflammatory actions in 3T3-L1 adipocytes