Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis

Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3β (Gsk-3β). Inhibition of Gsk-3β by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression.     

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.     

Cutting edge: precursor frequency affects the helper dependence of cytotoxic T cells.

Generation of CTL immunity often depends on the availability of CD4 T cell help. In this report, we show that CTL responses induced by cross-priming can be converted from CD4-dependent to CD4-independent by increasing the frequency of CTL precursors. In the absence of CD4 T cells, high numbers of CTL precursors were able to expand in number and become effector CTL. The ability of high frequencies of CD8 T cells to override help was not due to their ability to signal CD40 via expression of CD154. These findings suggest that when precursor frequencies are high, priming of CD8 T cell responses may not require CD4 T cell help.     

Sexual Dimorphism of the Feto-Placental Phenotype in Response to a High Fat and Control Maternal Diets in a Rabbit Model

Maternal environment during early developmental stages plays a seminal role in the establishment of adult phenotype. Using a rabbit model, we previously showed that feeding dams with a diet supplemented with 8% fat and 0.2% cholesterol (HH diet) from the prepubertal period and throughout gestation induced metabolic syndrome in adult offspring. Here, we examined the effects of the HH diet on feto-placental phenotype at 28 days post-coïtum (term = 31days) in relation to earlier effects in the blastocyst (Day 6). At 28 days, both male and female HH fetuses were intrauterine growth retarded and dyslipidemic, with males more affected than females. Lipid droplets accumulated in the HH placentas’ trophoblast, consistent with the increased concentrations in cholesteryl esters (3.2-fold), triacylglycerol (2.5-fold) and stored FA (2.12-fold). Stored FA concentrations were significantly higher in female compared to male HH placentas (2.18-fold, p<0.01), whereas triacylglycerol was increased only in HH males. Trophoblastic lipid droplet accumulation was also observed at the blastocyst stage. The expression of numerous genes involved in lipid pathways differed significantly according to diet both in term placenta and at the blastocyst stage. Among them, the expression of LXR-α in HH placentas was reduced in HH males but not females. These data demonstrate that maternal HH diet affects the blastocyst and induces sex-dependent metabolic adaptations in the placenta, which appears to protect female fetuses from developing severe dyslipidemia.     

The impact of introduced ship rats (Rattus rattus) on seedling recruitment and distribution of a subantarctic megaherb (Pleurophyllum hookeri)

Abstract The impact of introduced ship rats (Rattus rattus) on recruitment of the megaherb Pleurophyllum hookeri Buchan. (Asteraceae) was examined on subantarctic Macquarie Island, an island with no extant native terrestrial vertebrates. Pleurophyllum hookeri (Asteraceae) forms a dominant component of the Macquarie Island vegetation and is restricted to the subantarctic. The Macquarie Island population of P. hookeri is the most extensive and intact. Introduced ship rats (Rattus rattus) are well established in tall tussock grassland of Macquarie Island. We detected rat activity for the first time within P. hookeri herbfields, in autumn 2000. We found rats were destroying up to 90% of racemes. By excluding rats from caches of inflorescences that they had formed, we found they were having a significant negative effect on initial recruitment and seedling survival within the caches. However, because of high seedling mortality after 1 year, there was no sustained impact of the exclosures on P. hookeri seedling density.     

Spatial variation in the ongoing and widespread decline of a keystone plant species

Extensive dieback in dominant plant species in response to climate change is increasingly common. Climatic conditions and related variables, such as evapotranspiration, vary in response to topographical complexity. This complexity plays an important role in the provision of climate refugia. In 2008/2009, an island‐wide dieback event of the keystone cushion plant Azorella macquariensis Orchard (Apiaceae) occurred on sub‐Antarctic Macquarie Island. This signalled the start of a potential regime shift, suggested to be driven by increasing vapour pressure deficit. Eight years later, we quantified cover and dieback across the range of putative microclimates to which the species is exposed, with the aim of explaining dieback patterns. We test for the influence of evapotranspiration using a suite of topographic proxies and other variables as proposed drivers of change. We found higher cover and lower dieback towards the south of the island. The high spatial variation in A. macquariensis populations was best explained by latitude, likely a proxy for macroscale climate gradients and geology. Dieback was best explained by A. macquariensis cover and latitude, increasing with cover and towards the north of the island. The effect sizes of terrain variables that influence evapotranspiration rates were small. Island‐wide dieback remains conspicuous. Comparison between a subset of sites and historical data revealed a reduction of cover in the north and central regions of the island, and a shift south in the most active areas of dieback. Dieback remained comparatively low in the south. The presence of seedlings was independent of dieback. This study provides an empirical baseline for spatial variation in the cover and condition of A. macquariensis, both key variables for monitoring condition and ‘cover‐debt’ in this critically endangered endemic plant species. These findings have broader implications for understanding the responses of fellfield ecosystems and other Azorella species across the sub‐Antarctic under future climates.     

Systematic Dissection of the Metabolic-Apoptotic Interface in AML Reveals Heme Biosynthesis to Be a Regulator of Drug Sensitivity

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