SREBP1 is required for the induction by glucose of pancreatic beta-cell genes involved in glucose sensing

Previous studies have reported both positive and negative effects of culture of islets at high glucose concentrations on regulated insulin secretion. Here, we have reexamined this question in mouse islets and determined the role of changes in lipid synthesis in the effects of glucose. Glucose-stimulated insulin secretion (GSIS) and gene expression were examined in islets from C57BL/6 mice or littermates deleted for sterol-regulatory element binding protein-1 (SREBP1) after 4 days of culture at high glucose concentrations. Culture of control islets at 30 versus 8 mmol/l glucose led to enhanced secretion at both basal (3 mmol/l) and stimulatory (17 mmol/l) glucose concentrations and to enhanced triacylglycerol accumulation. These changes were associated with increases in the expression of genes involved in glucose sensing (glucose transporter 2, glucokinase, sulfonylurea receptor 1, inwardly rectifying K(+) channel 6.2), differentiation (pancreatic duodenal homeobox 1), and lipogenesis (Srebp1, fatty acid synthase, acetyl-coenzyme A carboxylase 1, stearoyl-coenzyme A desaturase 1). When cultured at either 8 or 30 mmol/l glucose, SREBP1-deficient (SREBP1(-/-)) islets displayed reduced GSIS and triacylglycerol content compared with normal islets. Correspondingly, glucose induction of the above genes in control islets was no longer observed in SREBP1(-/-) mouse islets. We conclude that enhanced lipid synthesis mediated by SREBP1c-dependent genes is required for the adaptive changes in islet gene expression and insulin secretion at high glucose concentrations.     

pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxidase: effect on schizophrenia susceptibility

Human genes coding for pLG72 and d-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein d-amino acid oxidase (hDAAO). In the brain this oxidizes d-serine, a potent activator of N-methyl-d-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of d-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of d-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility.     

Potassium Channel Silencing by Constitutive Endocytosis and Intracellular Sequestration

Tandem of P domains in a weak inwardly rectifying K⁺ channel 1 (TWIK1) is a K⁺ channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT₁R or adrenoreceptor α2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G_i protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.     

Effects of drought and elevated temperature on biochemical composition of forage plants and their impact on carbon storage in grassland soil

Aims

The objective of this study was to investigate the effects of future warming and drought on (1) the biochemical composition of above-ground biomass of forage plants (Festuca arundinacea and Dactylis glomerata), (2) the potential mineralization of this material in soil, and (3) its priming effect on native soil organic matter.

Methods

We sampled above-ground plant material from spring regrowth and summer regrowth of a climate change experiment. While in spring, the plants were well watered, the summer regrowth was exposed to drought and elevated temperature (+3 °C) by infrared heating of the canopy during 3 weeks. We assessed the elemental and isotopic composition, lignin and non-cellulosic carbohydrate content and composition of plant material grown under all three conditions. Its mineralization potential in soil and priming effects were evaluated during laboratory incubation.

Results

Warming had no significant effect on elemental and stable isotope composition of both plant materials. In contrast, it resulted in reduction of lignin content for both plant species and decrease of the lignin-to-N ratio for F. arundinacea and increased non-cellulosic carbohydrate content for D. glomerata. Summer regrowth was characterised by increase of δ¹³C values, which is consistent with variations in stomatal conductance due to water shortage. Moreover, summer drought induced an increase in N content leading to decrease of the C/N ratio and increase of lignin-to-N ratio of summer regrowth compared to spring regrowth. Differences in decomposition were small, while priming effects were more strongly altered by the different exposure to enviromental.

Conclusion

Our results provide direct experimental evidence that extreme climatic events (high temperature and precipitation deficit) have an influence on soil carbon storage particularly through their effect on priming of native soil organic matter induced by altered plant litter. These effects seem to be governed by alterations of stoichiometry and to a smaller extent by alterations of plant chemical composition.     

Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.     

Time and amplitude regulation of pulsatile insulin secretion by triggering and amplifying pathways in mouse islets

Glucose-induced insulin secretion is pulsatile. We investigated how the triggering pathway (rise in β-cell [Ca²⁺]_i) and amplifying pathway (greater Ca²⁺ efficacy on exocytosis) influence this pulsatility. Repetitive [Ca²⁺]_i pulses were imposed by high K⁺+ diazoxide in single mouse islets. Insulin secretion (measured simultaneously) tightly followed [Ca²⁺]_i changes. Lengthening [Ca²⁺]_i pulses increased the duration but not the amplitude of insulin pulses. Increasing glucose (5–20 mmol/l) augmented the amplitude of insulin pulses without changing that of [Ca²⁺]_i pulses. Larger [Ca²⁺]_i pulses augmented the amplitude of insulin pulses at high, but not low glucose. In conclusion, the amplification pathway ensures amplitude modulation of insulin pulses whose time modulation is achieved by the triggering pathway.     

ParaDB: a tool for paralogy mapping in vertebrate genomes

We present ParaDB (http://abi.marseille.inserm.fr/paradb/), a new database for large-scale paralogy studies in vertebrate genomes. We intended to collect all information (sequence, mapping and phylogenetic data) needed to map and detect new paralogous regions, previously defined as Paralogons. The AceDB database software was used to generate graphical objects and to organize data. General data were automatically collated from public sources (Ensembl, GadFly and RefSeq). ParaDB provides access to data derived from whole genome sequences (Homo sapiens, Mus musculus and Drosophila melanogaster): cDNA and protein sequences, positional information, bibliographical links. In addition, we provide BLAST results for each protein sequence, InParanoid orthologs and 'In-Paralogs' data, previously established paralogy data, and, to compare vertebrates and Drosophila, orthology data.     

Agonist-induced up-regulation of human somatostatin receptor type 1 is regulated by beta-arrestin-1 and requires an essential serine residue in the receptor C-tail

We have previously shown that the human somatostatin receptor type 1 (hSSTR1) does not undergo agonist-induced internalization, but is instead up-regulated at the membrane upon prolonged somatostatin (SST) exposure. The deletion of the carboxyterminal C-tail of the receptor completely abolishes up-regulation. To identify molecular signals that mediate hSSTR1 up-regulation, we created mutant receptors with progressive C-tail deletions. Up-regulation was found to be absent in mutants lacking residues Lys359-Ser360-Arg361. Moreover, point mutation of Ser360 to Ala completely abolished up-regulation. The coexpression of wild type hSSTR1 with V53D, a dominant negative mutant of beta-arrestin-1, completely blocked hSSTR1 up-regulation. Further analysis demonstrated that calcium-calmodulin (CaM) dependent kinases were essential for the SST-induced up-regulation response. Like wild type receptors, all mutants failed to internalize after agonist exposure and were able to inhibit forskolin-stimulated cAMP accumulation. Taking these data together, we suggest that SST-induced hSSTR1 up-regulation is critically dependent upon a specific Lys-Ser-Arg sequence in the C-tail of the receptor, with Ser360 being essential. Up-regulation also requires the participation of CaM protein kinases and interactions with beta-arrestins. In contrast, coupling to adenyl cyclase (AC) and internalization occur independently of molecular signals in the receptor's C-tail.