Increased lipid accumulation and insulin resistance in transgenic mice expressing DGAT2 in glycolytic (type II) muscle

Insulin resistance and type 2 diabetes are frequently accompanied by lipid accumulation in skeletal muscle. However, it is unknown whether primary lipid deposition in skeletal muscle is sufficient to cause insulin resistance or whether the type of muscle fiber, oxidative or glycolytic fiber, is an important determinant of lipid-mediated insulin resistance. Here we utilized transgenic mice to test the hypothesis that lipid accumulation specifically in glycolytic muscle promotes insulin resistance. Overexpression of DGAT2, which encodes an acyl-CoA:diacylglycerol acyltransferase that catalyzes triacylglycerol (TG) synthesis, in glycolytic muscle of mice increased the content of TG, ceramides, and unsaturated long-chain fatty acyl-CoAs in young adult mice. This lipid accumulation was accompanied by impaired insulin signaling and insulin-mediated glucose uptake in glycolytic muscle and impaired whole body glucose and insulin tolerance. We conclude that DGAT2-mediated lipid deposition specifically in glycolytic muscle promotes insulin resistance in this tissue and may contribute to the development of diabetes.     

Altered interaction and expression of proteins involved in neurosecretion in scrapie-infected GT1-1 cells

Prions cause transmissible and fatal diseases that are associated with spongiform degeneration, astrogliosis, and loss of axon terminals in the brains. To determine the expression of proteins involved in neurosecretion and synaptic functions after prion infection, gonadotropin-releasing hormone neuronal cell line subclone (GT1-1) was infected with the RML scrapie strain and analyzed by Western blotting, real time PCR, and immunohistochemistry. As revealed by Western blotting of lysates exposed to different temperatures, the levels of complexed SNAP-25, syntaxin 1A, and synaptophysin were decreased in scrapie-infected GT1-1 cells (ScGT1-1), whereas the level of monomeric forms of these proteins was increased and correlated to the level of scrapie prion protein (PrPSc). However, when complex formation was prevented by prolonged heating of samples in SDS, the levels of monomeric SNAP-25, syntaxin 1A and synaptophysin in ScGT1-1 cells were decreased in comparison to GT1-1 cells. The reduced level of SNAP-25 was observed as early as 32 days postinfection. Increased mRNA levels of both splice variants SNAP-25a and -b in ScGT1-1 cells were seen. No difference in the morphology, neuritic outgrowth or distribution of SNAP-25, syntaxin 1A, or synaptophysin could be observed in ScGT1-1 cells. Treatment with quinacrine or pentosan polysulfate cleared the PrPSc from the ScGT1-1 cell cultures, and the increase in levels of monomeric SNAP-25 and synaptophysin was reversible. These results indicate that a scrapie infection can cause changes in the expression of proteins involved in neuronal secretion, which may be of pathogenetic relevance for the axon terminal changes seen in prion-infected brains.     

Decline in lichen biodiversity on oak trunks due to urbanization

Biodiversity often suffers from urbanization. In the present study, we focused on how the duration of urbanization affects the richness of 17 epiphytic lichen species and their cover on large oaks in urban environments in a city of 100 000 inhabitants in southeast Sweden. We also surveyed trees in adjacent rural areas, selected to have similar distributions of tree trunk circumference and surrounding oak density (within 300 m). Lichen richness and cover were lower on urban trees compared to rural trees. Furthermore, richness and cover decreased with the length of time that urban trees had been surrounded by houses. Most of the species that were analysed demonstrated a decline in occurrence with respect to the duration of housing development. The reduction in the probability of occurrence varied from 60% (Calicium viride, Evernia prunastri), 80% (Chrysothrix candelaris) to 90% (Ramalina spp.) during the considered 160‐year period of urbanization. Therefore, even if valuable trees survive over the course of development, their lichen biota is likely to become depleted over time.     

Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside-out (cytoplasmic side-out) plasma membrane vesicles

Most of the plasma membrane vesicles formed upon homogenization of plant tissue have a right-side-out (cytoplasmic side-in) orientation. Subsequent purification of plasma membrane vesicles using aqueous two-phase partitioning leads to a further enrichment in right-side-out vesicles resulting in preparations with 80–90% of the vesicles in this orientation. Thus, to be able to assay, e.g. the ion-pumping activities of the H⁺-ATPase and the Ca²⁺-ATPase, which expose their active sites towards the cytoplasm, the vesicles have to be inverted. This is very efficiently achieved by including 0.05% of the detergent Brij 58 (C₁₆E₂₀) in the assay medium, which produces 100% sealed, inside-out (cytoplasmic side-out) vesicles from preparations of 80–90% right-side-out vesicles. This was shown by assaying ATP-dependent H⁺ pumping using the ΔpH probe acridine orange and dissipating the H⁺ gradient with nigericin, and by assaying ATP-dependent Ca²⁺ transport using ⁴⁵CA²⁺ and dissipating the Ca²⁺ gradient with the ionophore A23187. The presence of intact vesicles was confirmed by electronmicroscopy. The detergent Brij 58 is a polyoxyethylene acyl ether and a survey among some other members of this series revealed that those with a head group of relatively large size (E_20–23) showed this 'non-detergent behavior', whereas those with smaller head groups (E_8–10) behaved as normal detergents and permeabilized the membranes. Thus, a very convenient system for studies on ion-pumping activities and other vectorial properties of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.     

Lipids as cofactors in protein folding: stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells)

Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo alpha-lactalbumin and form HAMLET (human alpha-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein-fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo alpha-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid-protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process.     

Novel quantitative trait loci controlling development of experimental autoimmune encephalomyelitis and proportion of lymphocyte subpopulations

The B10.RIII mouse strain (H-2(r)) develops chronic experimental autoimmune encephalomyelitis (EAE) upon immunization with the myelin basic protein 89-101 peptide. EAE was induced and studied in a backcross between B10.RIII and the EAE-resistant RIIIS/J strain (H-2(r)), and a complete genome scan with microsatellite markers was performed. Five loci were significantly linked to different traits and clinical subtypes of EAE on chromosomes 1, 5, 11, 15, and 16, three of the loci having sex specificity. The quantitative trait locus on chromosome 15 partly overlapped with the Eae2 locus, previously identified in crosses between the B10.RIII and RIIIS/J mouse strains. The loci on chromosomes 11 and 16 overlapped with Eae loci identified in other mouse crosses. By analyzing the backcross animals for lymphocyte phenotypes, the proportion of B and T cells in addition to the levels of CD4(+)CD8(-) and CD4(-)CD8(+) T cells and the CD4(+)/CD8(+) ratio in spleen were linked to different loci on chromosomes 1, 2, 3, 5, 6, 11, and 15. On chromosome 16, we found significant linkage to spleen cell proliferation. Several linkages overlapped with the quantitative trait loci for disease phenotypes. The identification of subphenotypes that are linked to the same loci as disease traits could be most useful in the search for candidate genes and biological pathways involved in the pathological process.     

Neuronal α‐amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology

Recent studies indicate a crucial role for neuronal glycogen storage and degradation in memory formation. We have previously identified alpha‐amylase (α‐amylase), a glycogen degradation enzyme, located within synaptic‐like structures in CA1 pyramidal neurons and shown that individuals with a high copy number variation of α‐amylase perform better on the episodic memory test. We reported that neuronal α‐amylase was absent in patients with Alzheimer''s disease (AD) and that this loss corresponded to increased AD pathology. In the current study, we verified these findings in a larger patient cohort and determined a similar reduction in α‐amylase immunoreactivity in the molecular layer of hippocampus in AD patients. Next, we demonstrated reduced α‐amylase concentrations in oligomer amyloid beta 42 (Aβ₄₂) stimulated SH‐SY5Y cells and neurons derived from human‐induced pluripotent stem cells (hiPSC) with PSEN1 mutation. Reduction of α‐amylase production and activity, induced by siRNA and α‐amylase inhibitor Tendamistat, respectively, was further shown to enhance glycogen load in SH‐SY5Y cells. Both oligomer Aβ₄₂ stimulated SH‐SY5Y cells and hiPSC neurons with PSEN1 mutation showed, however, reduced load of glycogen. Finally, we demonstrate the presence of α‐amylase within synapses of isolated primary neurons and show that inhibition of α‐amylase activity with Tendamistat alters neuronal activity measured by calcium imaging. In view of these findings, we hypothesize that α‐amylase has a glycogen degrading function within synapses, potentially important in memory formation. Hence, a loss of α‐amylase, which can be induced by Aβ pathology, may in part underlie the disrupted memory formation seen in AD patients.     

Gab2 is involved in differential phosphoinositide 3-kinase signaling by two splice forms of c-Kit

The stem cell factor receptor/c-Kit plays an important physiological role in hematopoiesis, melanogenesis, and gametogenesis. It has also been implicated in numerous human malignancies. Signal transduction pathways shown to be of importance for c-Kit-mediated transformation include the phosphoinositide 3-kinase (PI3K)/Akt pathway. We have previously shown that two alternative splice forms of c-Kit, denoted GNNK(-) and GNNK(+), mediate distinctively different signals. In this study, we found that in the hematopoietic cell line Ba/F3, GNNK(-) c-Kit mediates a substantially stronger activation of PI3K/Akt than GNNK(+) c-Kit. This difference in signaling was shown to be dependent on the association of the scaffolding protein Gab2 with c-Kit, and Src-mediated phosphorylation of Gab2 was shown to be to be independent of the direct association of PI3K with c-Kit. Furthermore, proliferation and survival of Ba/F3 cells expressing a mutant of c-Kit that fails to bind to PI3K directly were slightly decreased compared with wild-type c-Kit-expressing cells. Using small interfering RNA technology, we further verified a role of Gab2 in inducing activation of PI3K/Akt downstream of c-Kit. To summarize, we show that PI3K activation by c-Kit is both splice form-dependent and cell type-specific. Furthermore, activation of PI3K by c-Kit is dependent both on the direct PI3K-binding site in c-Kit and on the phosphorylation of Gab2. The fact that c-Kit has been found mutated in numerous human malignancies, including acute myeloid leukemia, and that Gab2 is often overexpressed in acute myeloid leukemia suggests a potential role of Gab2-mediated PI3K activation in transformation.