Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.     

Sample preparation and analytical strategies for large-scale phosphoproteomics experiments

Reversible protein phosphorylation is an important post-translational modification that controls a wide range of protein functions including enzyme activity, subcellular localisation, protein degradation, intra- and inter-molecular protein interactions. Significant advances in both phosphopeptide enrichment methods and sensitive mass spectrometry instrumentation have been achieved over the past decade to facilitate the large-scale identification of protein phosphorylation in humans and different animal and microbial model systems. While mass spectrometry provides the ability to identify thousands of phosphorylation sites in a single experiment, the further understanding of the functional significance of this modification on protein substrates requires detailed information on the changes in phosphorylation stoichiometry and protein abundance across experimental paradigms. This review presents different sample preparation methods and analytical strategies used in mass spectrometry-based phosphoproteomics to profile protein phosphorylation and unravel the regulation of this modification on protein function.     

Tracing the origin of disjunct distributions: a case of biogeographical convergence in Pyrgus butterflies

Aim To study the biogeographical factors responsible for the current disjunct distributions of two closely related species of butterflies (Pyrgus cinarae and Pyrgus sidae, Lepidoptera: Hesperioidea). Both species have small populations in the Iberian Peninsula that are isolated by more than 1000 km from their nearest conspecifics. Because these species possess similar ecological preferences and geographical distributions, they are excellent candidates for congruent biogeographical histories. Location The Palaearctic region, with a special focus on the Mediterranean peninsulas as glacial refugia. Methods We integrated phylogeography and population genetic analyses with ecological niche modelling. The mitochondrial gene cytochrome c oxidase subunit 1 (COI) and the non‐coding nuclear marker internal transcribed spacer 2 (ITS2) were analysed for 62 specimens of P. cinarae and for 80 of P. sidae to infer phylogeography and to date the origin of disjunct distributions. Current and ancestral [Last Glacial Maximum using MIROC (Model for Interdisciplinary Research on Climate) and CCSM (Community Climate System Model) circulation models] distribution models were calculated with Maxent . Using present climatic conditions, we delimited the ecological space for each species. Results The genetic structure and potential ancestral distribution of the two species were markedly different. While the Iberian population of P. cinarae had an old origin (c. 1 Ma), that of P. sidae was closely related to French and Italian lineages (which jointly diverged from eastern populations c. 0.27 Ma). Ecological niche modelling showed that minor differences in the ecological preferences of the two species seem to account for their drastically different distributional response to the last glacial to post‐glacial environmental conditions. Although the potential distribution of P. cinarae was largely unaffected by climate change, suitable habitat for P. sidae strongly shifted in both elevation and latitude. This result might explain the early origin of the disjunct distribution of P. cinarae, in contrast to the more recent disjunction of P. sidae. Main conclusions We show that convergent biogeographical patterns can be analysed with a combination of genetic and ecological niche modelling data. The results demonstrate that species with similar distributional patterns and ecology may still have different biogeographical histories, highlighting the importance of including the temporal dimension when studying biogeographical patterns.     

Genetic polymorphisms potentially associated with response to metformin in postmenopausal diabetics suffering and not suffering with cancer

Metformin is a well-known antidiabetic medication, which, besides diabetes, may be involved into modulation of other age-related pathologies, including cancer. The study concerns 12 gene polymorphisms divided into 2 groups consisting of 6 genes each. The first group was composed from so-called “standard” (S) polymorphisms, for which the connection with metabolic response to metformin is already established. The second group included polymorphisms of genes encoding proteins possibly connected with diabetes mellitus type 2 (DM2), impaired glucose tolerance or cancer and entitled here as “associated” (A). A total of 156 postmenopausal women (average age 60.7 ± 0.7) were included, 37 of them healthy, 64 with type DM2 and concurrent treatment-naïve cancer (mostly breast, endometrial or colorectal cancer), 32 with DM2 without cancer, and 23 with treatment-naïve cancer and normal glucose tolerance. The leading metformin response S-marker in combined group of DM2 patients was the CC variant of OCT1-R61C polymorphism of organic cation transporter protein 1 gene. In cancer patients without DM2, this position belonged to AC and AA genotypes of OCT1_rs622342 polymorphism. Among the A-polymorphisms, GA variant of sex hormone-binding globulin gene SHBG_D356N was less frequently observed in DM2 patients with or without cancer. Besides, in diabetics, the same polymorphic variant of SHBG as well as GC genotype of oxidized lipoprotein receptor OLR1_G501C and GG genotype of locus rs11065987 near BRAP gene were carried rather often in combination with “metformin-positive” variant of OCT1_R61C. In addition, carriers of OCT1_R61C and OCT1_rs622342 polymorphisms with potentially positive reaction to metformin had higher insulin resistance score (HOMA-IR) values. Received data lead to the conclusion that postmenopausal diabetics, both with and without cancer, differ in genetic stigmata of potential response to metformin less than they differ from cancer patients without DM2. As genetic polymorphisms associated with metabolic and anticancer metformin (and, possibly, phenformin) effects may be different, this subject requires further investigation.     

Multisite phosphorylation of the NDC80 complex gradually tunes its microtubule-binding affinity

Microtubule (MT) attachment to kinetochores is vitally important for cell division, but how these interactions are controlled by phosphorylation is not well known. We used quantitative approaches in vitro combined with molecular dynamics simulations to examine phosphoregulation of the NDC80 complex, a core kinetochore component. We show that the outputs from multiple phosphorylation events on the unstructured tail of its Hec1 subunit are additively integrated to elicit gradual tuning of NDC80-MT binding both in vitro and in silico. Conformational plasticity of the Hec1 tail enables it to serve as a phosphorylation-controlled rheostat, providing a new paradigm for regulating the affinity of MT binders. We also show that cooperativity of NDC80 interactions is weak and is unaffected by NDC80 phosphorylation. This in vitro finding strongly supports our model that independent molecular binding events to MTs by individual NDC80 complexes, rather than their structured oligomers, regulate the dynamics and stability of kinetochore-MT attachments in dividing cells.     

Migratory routes of red‐necked phalaropes Phalaropus lobatus breeding in southern Chukotka revealed by geolocators

The migration routes of red‐necked phalaropes breeding around the Bering Sea are poorly known, despite the fact that the Bering Sea could mark the boundary between the East Palearctic populations that winter in the Pacific Ocean around the East Indies and the West Nearctic populations that winter in the Pacific Ocean off the coast of South America. Geolocator data retrieved from two male phalaropes tagged in southern Chukotka, Far Eastern Russia, confirm that birds breeding in this region belong to the East Palearctic population and winter in the East Indies, suggesting that the division line with the West Nearctic population is farther to the east. The routes taken by the two phalaropes were almost entirely pelagic, totaling around 18 000–20 000 km round‐trip, with the birds continuously on the move during migration, rather than resident in any particular stopover site, contrary to most other migratory shorebirds.