Rho-type GTPases control many cytoskeletal rearrangements, but their regulation remains poorly understood. Here, we show that in S. cerevisiae, activation of the CDK Cdc28-Cln2 at bud emergence triggers relocalization of Cdc24, the GEF for Cdc42, from the nucleus to the polarization site, where it is stably maintained by binding to the adaptor Bem1. Locally activated Cdc42 then polarizes the cytoskeleton in a manner dependent on its effectors Bni1 and the PAK-like kinase Cla4. In addition, Cla4 induces phosphorylation of Cdc24, leading to its dissociation from Bem1 at bud tips, thereby ending polarized bud growth in vivo. Our results thus suggest a dynamic temporal and spatial regulation of the Cdc42 module: Cdc28-Cln triggers actin polarization by activating Cdc42, which in turn restricts its own activation via a negative feedback loop acting on its GEF Cdc24.

Aims/hypothesis We explored the potential adverse effects of pro-atherogenic oxidised LDL-cholesterol particles on beta cell function. Materials and methods Isolated human and rat islets and different insulin-secreting cell lines were incubated with human oxidised LDL with or without HDL particles. The insulin level was monitored by ELISA, real-time PCR and a rat insulin promoter construct linked to luciferase gene reporter. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. Results Prolonged incubation with human oxidised LDL particles led to a reduction in preproinsulin expression levels, whereas the insulin level was preserved in the presence of native LDL-cholesterol. The loss of insulin production occurred at the transcriptional levels and was associated with an increase in activator protein-1 transcriptional activity. The rise in activator protein-1 activity resulted from activation of c-Jun N-terminal kinases (JNK, now known as mitogen-activated protein kinase 8 [MAPK8]) due to a subsequent decrease in islet-brain 1 (IB1; now known as MAPK8 interacting protein 1) levels. Consistent with the pro-apoptotic role of the JNK pathway, oxidised LDL also induced a twofold increase in the rate of beta cell apoptosis. Treatment of the cells with JNK inhibitor peptides or HDL countered the effects mediated by oxidised LDL. Conclusions/interpretation These data provide strong evidence that oxidised LDL particles exert deleterious effects in the progression of beta cell failure in diabetes and that these effects can be countered by HDL particles.

OBJECTIVE— The pro-inflammatory cytokine interleukin-1β (IL-1β) generates pancreatic β-cells apoptosis mainly through activation of the c-Jun NH2-terminal kinase (JNK) pathway. This study was designed to investigate whether the long-acting agonist of the hormone glucagon-like peptide 1 (GLP-1) receptor exendin-4 (ex-4), which mediates protective effects against cytokine-induced β-cell apoptosis, could interfere with the JNK pathway. RESEARCH DESIGN AND METHODS— Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1β. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS— Ex-4 inhibited induction of the JNK pathway elicited by IL-1β. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1β. CONCLUSIONS— The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1β and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.

Islet-brain 1 (IB1) is the rat and human homologue of JIP-1, a murine inhibitor of the c-Jun amino-terminal kinase (JNK). It was termed IB1 since its expression is mostly detectable in pancreatic islets and in the brain (2). IB1 was identified by studying the transcriptional mechanisms responsible for the pancreatic ␤-cell-specific control of glucose transporter gene GLUT2 (2,4,24,36). Subsequently, the human MAPK8IP1 gene, encoding IB1, was established as a candidate gene for diabetes mellitus (35). Indeed, the direct sequencing of this gene in human type 2 diabetes patients revealed the presence of a missense mutation (resulting in protein mutation S59N) which cosegregated with a rare form of monogenic type 2 diabetes. Ex vivo, this mutation was shown to induce an accelerated apoptosis in pancreatic ␤ cells (35). These observations identified IB1 as a key regulator for ␤-cell survival since it modulates the activation of the JNK signaling pathway, a system which plays an essential role in maturation, differentiation, and/or apoptosis (8, 16). For example, when the IB1 protein content is decreased, ␤ cells are more sensitive to cytokineinduced apoptosis by increasing the JNK activity (3). Thus, the IB1 expression level is critical for ␤-cell function.The goal of the present study was to understand how MAPK8IP1 gene expression is controlled in a tissue-specific manner. Work by Atouf and coauthors has correlated the selective presence of several gene transcripts in pancreatic ␤ and neuronal cells with the absence in these cells of a transcription factor named REST (RE-1 silencing transcription factor, also termed as NRSF) (1). This protein is a zinc finger transcriptional repressor found to be widely expressed during embryogenesis in all tissues, except in endocrine pancreas and mature neuronal tissues (1, 5). The REST gene displays modular organization conserved across humans, rats, and mice within the protein-coding region and is regulated by alternative splicing of REST pre-mRNA. It was reported that the REST isoform with nine zinc fingers is the most predominant isoform found in various tissues (25). Alternatively spliced REST protein isoforms differ in their DNA-binding domains and transrepression domains (26), suggesting different functions of REST. For example, the REST4 isoform, which is a truncated protein, inhibits REST activity by acting as a dominant-negative form of REST (DNREST) (26,32). REST binds to a 21-bp cis element called the RE-1 silencer element (NRSE), also known as repressor element RE-1, to negatively regulate in nonneuronal tissues several genes preferentially expressed in neuronal cells such as the rat SCG10, the rat type II sodium channel, the human synapsin I, and the rat N-methyl-D-aspartate (NMDA) receptor 1 genes (6,18,20,33). The repression effect induced by REST required the interaction of REST with the corepressor mSin3 and histone deacetylase I (HDACI) to form a complex which induces hypoacetylation of histone (15). These authors proposed that a remodeling of the chromatin stru...

Aims/hypothesisPro-atherogenic and pro-oxidant, oxidised LDL trigger adverse effects on pancreatic beta cells, possibly contributing to diabetes progression. Because oxidised LDL diminish the expression of genes regulated by the inducible cAMP early repressor (ICER), we investigated the involvement of this transcription factor and of oxidative stress in beta cell failure elicited by oxidised LDL.MethodsIsolated human and rat islets, and insulin-secreting cells were cultured with human native or oxidised LDL or with hydrogen peroxide. The expression of genes was determined by quantitative real-time PCR and western blotting. Insulin secretion was monitored by EIA kit. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei.ResultsExposure of beta cell lines and islets to oxidised LDL, but not to native LDL raised the abundance of ICER. Induction of this repressor by the modified LDL compromised the expression of important beta cell genes, including insulin and anti-apoptotic islet brain 1, as well as of genes coding for key components of the secretory machinery. This led to hampering of insulin production and secretion, and of cell survival. Silencing of this transcription factor by RNA interference restored the expression of its target genes and alleviated beta cell dysfunction and death triggered by oxidised LDL. Induction of ICER was stimulated by oxidative stress, whereas antioxidant treatment with N-acetylcysteine or HDL prevented the rise of ICER elicited by oxidised LDL and restored beta cell functions.Conclusions/interpretationInduction of ICER links oxidative stress to beta cell failure caused by oxidised LDL and can be effectively abrogated by antioxidant treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2165-x) contains supplementary material, which is available to authorised users.

The neuronal-specific protein complexin I (CPX I) plays an important role in controlling the Ca2+-dependent neurotransmitter release. Since insulin exocytosis and neurotransmitter release rely on similar molecular mechanisms and that pancreatic β-cells and neuronal cells share the expression of many restricted genes, we investigated the potential role of CPX I in insulin-secreting cells. We found that pancreatic islets and several insulin-secreting cell lines express high levels of CPX I. The β-cell expression of CPX I is mediated by the presence of a neuron restrictive silencer element located within the regulatory region of the gene. This element bound the transcriptional repressor REST, which is found in most cell types with the exception of mature neuronal cells and β-cells. Overexpression of CPX I or silencing of the CPX I gene (Cplx1) by RNA interference led to strong impairment in β-cell secretion in response to nutrients such as glucose, leucine and KCl. This effect was detected both in the early and the sustained secretory phases but was much more pronounced in the early phase. We conclude that CPX I plays a critical role in β-cells in the control of the stimulated-exocytosis of insulin.

The terminal differentiation of neuronal and pancreatic ␤-cells requires the specific expression of genes that are targets of an important transcriptional repressor named RE-1 silencing transcription factor (REST). The molecular mechanism by which these REST target genes are expressed only in neuronal and ␤-cells and are repressed by REST in other tissues is a central issue in differentiation program of neuronal and ␤-cells. Herein, we showed that the transcriptional factor Sp1 was required for expression of most REST target genes both in insulin-secreting cells and neuronal-like cells where REST is absent. Inhibition of REST in a non-␤ and a non-neuronal cell model restored the transcriptional activity of Sp1. This activity was also restored by trichostatin A indicating the requirement of histone deacetylases for the REST-mediated silencing of Sp1. Conversely, exogenous introduction of REST blocked Sp1-mediated transcriptional activity. The REST inhibitory effect was mediated through its C-terminal repressor domain, which could interact with Sp1. Taken together, these data show that the inhibition of Sp1 by REST is required for the silencing of its target genes expression in non-neuronal and in non-␤-cells. We conclude that the interplay between REST and Sp1 determines the cell-specific expression of REST target genes.

The transcriptional repressor RE1 silencer transcription factor (REST) is an important factor that restricts some neuronal traits to neurons. Since these traits are also present in pancreatic beta-cells, we evaluated their role by generating a model of insulin-secreting cells that express REST. The presence of REST led to a decrease in expression of its known target genes, whereas insulin expression and its cellular content were conserved. As a consequence of REST expression, the capacity to secrete insulin in response to mitochondrial fuels, a particularity of mature beta-cells, was impaired. These data provide evidence that REST target genes are required for an appropriate glucose-induced insulin secretion.