Recombinant Adiponectin Does Not Lower Plasma Glucose in Animal Models of Type 2 Diabetes

Aims/Hypothesis

Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats.

Methods

Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast. The purified protein was administered at 10–30 mg/kg i.p. b.i.d. to diabetic db/db mice for 2 weeks. Furthermore, high molecular weight human and globular mouse adiponectin batches were administered at 5–15 mg/kg i.p. b.i.d. to diabetic sand rats for 12 days.

Results

Surprisingly, none of our batches had any effect on blood glucose, HbA1c, plasma lipids or body weight in diabetic db/db mice or sand rats. In vitro biological, biochemical and biophysical data suggest that the protein was correctly folded and biologically active.

Conclusions/Interpretation

Recombinant adiponectin is ineffective at lowering blood glucose in diabetic db/db mice or sand rats.     

Identification of new cell size control genes in <Emphasis Type="Italic">S. cerevisiae</Emphasis>

Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a “critical cell size” must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened an additional 779 yeast knockouts containing 435 new ORFs (~7% of the yeast genome) to supplement previous cell size screens. Upon completion, 10 new strong size mutants were identified: nine in log-phase cells and one in saturation-phase cells, and 97% of the yeast genome has now been screened for cell size mutations. The majority of the logarithmic phase size mutants have functions associated with translation further implicating the central role of growth control in the cell division process. Genetic analyses suggest ECM9 is directly associated with the START transition. Further, the small (whi) mutants mrpl49Δ and cbs1Δ are dependent on CLN3 for cell size effects. In depth analyses of new size mutants may facilitate a better understanding of the processes that govern cell size homeostasis.     

Structure and 3D Arrangement of Endoplasmic Reticulum Membrane-Associated Ribosomes

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Highlights? Subtomogram average of the canine ER-associated ribosome in situ at 31 Å resolution ? Large subunit rRNA ES27L is in direct contact with the ER membrane ? Sec61, TRAP, and potentially OST and the SP complex are resolved ? ER-associated ribosomes adopt a preferred arrangement, likely polyribosome specific     

Visualizing the genetic landscape of Arabidopsis seed performance

Perfect timing of germination is required to encounter optimal conditions for plant survival and is the result of a complex interaction between molecular processes, seed characteristics, and environmental cues. To detangle these processes, we made use of natural genetic variation present in an Arabidopsis (Arabidopsis thaliana) Bayreuth × Shahdara recombinant inbred line population. For a detailed analysis of the germination response, we characterized rate, uniformity, and maximum germination and discuss the added value of such precise measurements. The effects of after-ripening, stratification, and controlled deterioration as well as the effects of salt, mannitol, heat, cold, and abscisic acid (ABA) with and without cold stratification were analyzed for these germination characteristics. Seed morphology (size and length) of both dry and imbibed seeds was quantified by using image analysis. For the overwhelming amount of data produced in this study, we developed new approaches to perform and visualize high-throughput quantitative trait locus (QTL) analysis. We show correlation of trait data, (shared) QTL positions, and epistatic interactions. The detection of similar loci for different stresses indicates that, often, the molecular processes regulating environmental responses converge into similar pathways. Seven major QTL hotspots were confirmed using a heterogeneous inbred family approach. QTLs colocating with previously reported QTLs and well-characterized mutants are discussed. A new connection between dormancy, ABA, and a cripple mucilage formation due to a naturally occurring mutation in the MUCILAGE-MODIFIED2 gene is proposed, and this is an interesting lead for further research on the regulatory role of ABA in mucilage production and its multiple effects on germination parameters.     

An efficient transformation method for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.     

Triple mammalian/yeast/bacterial shuttle vectors for single and combined Lentivirus- and Sindbis virus-mediated infections of neurons

Today, a large variety of viral vectors is available for ectopic gene expression in mammalian cell cultures or in vivo. Among them, infection with Sindbis virus- or Lentivirus-derived constructs is often used to address biological questions or for applications in neuronal therapies. However, cloning of genes of interest is time consuming, since it relies on restriction and ligation, frequently of PCR-generated DNA fragments with suitable restriction sites introduced by the primers employed. We here take advantage of the unusually high capacity for homologous recombination in Saccharomyces cerevisiae to circumvent this problem, and introduce a new set of triple shuttle vectors, which can be shuffled between E. coli, yeast, and mammalian cells. The system allows the introduction of genes of interest largely independent of the target site in the vectors. It also allows the removal of the yeast selection marker by Cre-recombinase directed recombination in E. coli, if vector size limits transfection efficiency in the mammalian cells. We demonstrate the expression of genes encoding fluorescent proteins (EGFP and mCherry) both separately and in combination, using two different viral systems in mammalian cell lines, primary neurons and organotypic slices.     

Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk

Background

The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-??/??1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1?? induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells.

Results

Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1?? solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling.

Conclusions

Here we show that hybrid cells could evolve exhibiting a differential active RAF-AKT crosstalk. Because PI3K/AKT signalling has been chosen as a target for anti-cancer therapies our data might point to a possible severe side effect of AKT targeted cancer therapies. Inhibition of PI3K/AKT signalling in RAF-AKT crosstalk positive cancer (hybrid) cells could result in a progression of these cells. Thus, not only the receptor (activation) status, but also the activation of signal transduction molecules should be analysed thoroughly prior to therapy.     

Fasting goldfish, Carassius auratus, and common carp, Cyprinus carpio, use different metabolic strategies when swimming

Fish need to balance their energy use between digestion and other activities, and different metabolic compromises can be pursued. We examined the effects of fasting (7days) on metabolic strategies in goldfish and common carp at different swimming levels. Fasting had no significant effect on swimming performance (U(crit)) of either species. Feeding and swimming profoundly elevated total ammonia (T(amm)) excretion in both species. In fed goldfish, this resulted in increased ammonia quotients (AQ), and additionally plasma and tissue ammonia levels increased with swimming reflecting the importance of protein contribution for aerobic metabolism. In carp, AQ did not change since oxygen consumption (MO(2)) and T(amm) excretion followed the same trend. Plasma ammonia did not increase with swimming suggesting a balance between production and excretion rate except for fasted carp at U(crit). While both species relied on anaerobic metabolism during exhaustive swimming, carp also showed increased lactate levels during routine swimming. Fasting almost completely depleted glycogen stores in carp, but not in goldfish. Both species used liver protein for basal metabolism during fasting and muscle lipid during swimming. In goldfish, feeding metabolism was sacrificed to support swimming metabolism with similar MO(2) and U(crit) between fasted and fed fish, whereas in common carp feeding increased MO(2) at U(crit) to sustain feeding and swimming independently.