Ras inhibition induces insulin sensitivity and glucose uptake

Background

Reduced glucose uptake due to insulin resistance is a pivotal mechanism in the pathogenesis of type 2 diabetes. It is also associated with increased inflammation. Ras inhibition downregulates inflammation in various experimental models. The aim of this study was to examine the effect of Ras inhibition on insulin sensitivity and glucose uptake, as well as its influence on type 2 diabetes development.

Methods and Findings

The effect of Ras inhibition on glucose uptake was examined both in vitro and in vivo. Ras was inhibited in cells transfected with a dominant-negative form of Ras or by 5-fluoro-farnesylthiosalicylic acid (F-FTS), a small-molecule Ras inhibitor. The involvement of IκB and NF-κB in Ras-inhibited glucose uptake was investigated by immunoblotting. High fat (HF)-induced diabetic mice were treated with F-FTS to test the effect of Ras inhibition on induction of hyperglycemia. Each of the Ras-inhibitory modes resulted in increased glucose uptake, whether in insulin-resistant C2C12 myotubes in vitro or in HF-induced diabetic mice in vivo. Ras inhibition also caused increased IκB expression accompanied by decreased expression of NF-κB . In fat-induced diabetic mice treated daily with F-FTS, both the incidence of hyperglycemia and the levels of serum insulin were significantly decreased.

Conclusions

Inhibition of Ras apparently induces a state of heightened insulin sensitization both in vitro and in vivo. Ras inhibition should therefore be considered as an approach worth testing for the treatment of type 2 diabetes.     

Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells

Ras-membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras-lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins with nonraft sites, which may underlie functional differences and phenotypic variation between different Ras isoforms, are unexplored. Here we used lateral mobility studies by FRAP to investigate the membrane interactions of green fluorescent protein-tagged H- and K-Ras in live cells. All Ras isoforms displayed stable membrane association, moving by lateral diffusion and not by exchange with a cytoplasmic pool. The lateral diffusion rates of constitutively active K- and H-Ras increased with their expression levels in a saturable manner, suggesting dynamic association with saturable sites or domains. These sites are distinct from lipid rafts, as the activated Ras mutants are not raft resident. Moreover, they appear to be different for H- and K-Ras. However, wild-type H-Ras, the only isoform preferentially localized in rafts, displayed cholesterol-sensitive interactions with rafts that were independent of its expression level. Our findings provide a mechanism for selective signaling by different Ras isoforms.     

Rabbit EPO gene and cDNA: expression of rabbit EPO after intramuscular injection of pDNA

Erythropoietin (EPO) cDNA was cloned from kidney total RNA of a NZW rabbit. The cDNA comprises a 588-bp open reading frame encoding a 195 amino acid protein with distinguishable regions of high of homology to other mammalian EPOs. Intramuscular injection of mice with a rabbit EPO expression plasmid resulted in a significant hematocrit increase. A rabbit genomic DNA fragment was also cloned using the rabbit EPO cDNA. This 4312-bp genomic DNA fragment contains sequences homologous to the mouse EPO promoter and hypoxia-responsive enhancer. In addition, the genomic DNA also presents a high degree of conservation to other regions involved in hypoxia response. Sequence divergence in the 3' UTR may indicate differences in regulation of mRNA stability or response to low oxygen tension.     

Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity

Depending on the cellular context, Ras can activate characteristic effectors by mechanisms still poorly understood. Promotion by galectin-1 of Ras activation of Raf-1 but not of phosphoinositide 3-kinase (PI3-K) is one such mechanism. In this report, we describe a mechanism controlling selectivity of K-Ras4B (K-Ras), the most important Ras oncoprotein. We show that galectin-3 acts as a selective binding partner of activated K-Ras. Galectin-3 co-immunoprecipitated significantly better with K-Ras-GTP than with K-Ras-GDP, H-Ras, or N-Ras and colocalized with green fluorescent protein-K-Ras(G12V), not with green fluorescent protein-H-Ras(G12V), in the cell membrane. Co-transfectants of K-Ras/galectin-3, but not of H-Ras/galectin-3, exhibited enhanced and prolonged epidermal growth factor-stimulated increases in Ras-GTP, Raf-1 activity, and PI3-K activity. Extracellular signal-regulated kinase (ERK) activity, however, was attenuated in K-Ras/galectin-3 and in K-Ras(G12V)/galectin-3 co-transfectants. Galectin-3 antisense RNA inhibited the epidermal growth factor-stimulated increase in K-Ras-GTP but enhanced ERK activation and augmented K-Ras(G12V) transformation activity. Thus, unlike galectin-1, which prolongs Ras activation of ERK and inhibits PI3-K, K-Ras-GTP/galectin-3 interactions promote, in addition to PI3-K and Raf-1 activation, a third inhibitory signal that attenuates active ERK. These experiments established a novel and specific mechanism controlling the duration and selectivity of signals of active K-Ras, which is extremely important in many human tumors.     

Pathway- and expression level-dependent effects of oncogenic N-Ras: p27(Kip1) mislocalization by the Ral-GEF pathway and Erk-mediated interference with Smad signaling

Overactivation of Ras pathways contributes to oncogenesis and metastasis of epithelial cells in several ways, including interference with cell cycle regulation via the CDK inhibitor p27(Kip1) (p27) and disruption of transforming growth factor beta (TGF-beta) anti-proliferative activity. Here, we show that at high expression levels, constitutively active N-Ras induces cytoplasmic mislocalization of murine and human p27 via the Ral-GEF pathway and disrupts TGF-beta-mediated Smad nuclear translocation by activation of the Mek/Erk pathway. While human p27 could also be mislocalized via the phosphatidylinositol 3-kinase/Akt pathway, only Ral-GEF activation was effective for murine p27, which lacks the Thr157 Akt phosphorylation site of human p27. This establishes a novel role for the Ral-GEF pathway in regulating p27 localization. Interference with either Smad translocation or p27 nuclear localization was sufficient to disrupt TGF-beta growth inhibition. Moreover, expression of activated N-Ras or specific effector loop mutants at lower levels using retroviral vectors induced p27 mislocalization but did not inhibit Smad2/3 translocation, indicating that the effects on p27 localization occur at lower levels of activated Ras. These findings have important implications for the contribution of activated Ras to oncogenesis and for the conversion of TGF-beta from an inhibitory to a metastatic factor in some epithelial tumors.     

Individual palmitoyl residues serve distinct roles in H-ras trafficking, microlocalization, and signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.     

Successful pregnancies in cows following double freezing of a large volume of semen

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes. Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws. All freezing was done in a "Multi thermal gradient" (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) x (V). Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode. We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method. The results showed a 75 +/- 5% post thaw motility after freezing a 12 mL test tube and 50 +/- 5% after a second freezing/thawing in mini-straws, respectively. Controlled vapour freezing showed a 60 +/- 10% post thaw motility. The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 45.5% (56/123) for the controlled group. These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume.     

A comparative study of the lipid composition of yeasts with different fermentative capacities

Summary The lipid composition of a classical yeast and a poor fermenter, at low and high sugar concentrations, was compared. Polyunsaturated fatty acids (18:2, 18:3) were found in the osmotolerant weak fermenter, Saccharomyces mellis, their content decreasing with an increase of glucose levels, while the highly fermenting yeast S. cerevisiae had no polyunsaturated fatty acids at all sugar concentrations examined. Also total unsaturation of fatty acids ( mol^–1) was significantly higher with S. mellis. The sterol content varied considerably, being higher with the highly fermenting yeasts and low with S. mellis and the film yeast Pichia sp. The ratio of free sterols/phospholipids was high in S. cerevisiae (1:7) and low in S. mellis (1:177). Hybrid yeasts (S. cerevisiaexS. mellis) which were the best fermenting organisms in our study, also showed a high ratio of free sterols/phospholipids (1:6–1:8). A correlation between the fermentative capacity of yeasts and the fluidity of their membranes is suggested.