Dorothée Caille

Regulation of PKD by the MAPK p38δ in Insulin Secretion and Glucose Homeostasis

Summary Dysfunction and loss of insulin-producing pancreatic β cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38δ display improved glucose tolerance due to enhanced insulin secretion from pancreatic β cells. Deletion of p38δ results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38δ catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38δ null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated β cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38δ-deficient islets and glucose tolerance in p38δ null mice as well as their susceptibility to oxidative stress. In conclusion, the p38δ-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic β cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

Dietary phytoestrogens activate AMP-activated protein kinase with improvement in lipid and glucose metabolism

OBJECTIVE— Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents. RESEARCH DESIGN AND METHODS— Male CD-1 mice were fed from conception to adulthood with either a high soy–containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion. RESULTS— In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism. CONCLUSIONS— Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia by Pseudomonas aeruginosa

ABSTRACT The opportunistic bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis and immunocompromised individuals. Bacterial adherence to the basolateral domain of the host cells and internalization are thought to participate in P. aeruginosa pathogenicity. However, the mechanism by which the pathogen initially modulates the paracellular permeability of polarized respiratory epithelia remains to be understood. To investigate this mechanism, we have searched for virulence factors secreted by P. aeruginosa that affect the structure of human airway epithelium in the early stages of infection. We have found that only bacterial strains secreting rhamnolipids were efficient in modulating the barrier function of an in vitro-reconstituted human respiratory epithelium, irrespective of their release of elastase and lipopolysaccharide. In contrast to previous reports, we document that P. aeruginosa was not internalized by epithelial cells. We further report that purified rhamnolipids, applied on the surfaces of the epithelia, were sufficient to functionally disrupt the epithelia and to promote the paracellular invasion of rhamnolipid-deficient P. aeruginosa. The mechanism involves the incorporation of rhamnolipids within the host cell membrane, leading to tight-junction alterations. The study provides direct evidence for a hitherto unknown mechanism whereby the junction-dependent barrier of the respiratory epithelium is selectively altered by rhamnolipids.

Connexin30 deficiency causes instrastrial fluid–blood barrier disruption within the cochlear stria vascularis

The endocochlear potential (EP) is essential to hearing, because it provides approximately half of the driving force for the mechanoelectrical transduction current in auditory hair cells. The EP is produced by the stria vascularis (SV), a vascularized bilayer epithelium of the cochlea lateral wall. The absence of the gap junction protein connexin30 (Cx30) in Cx30−/− mice results in the SV failure to produce an EP, which mainly accounts for the severe congenital hearing impairment of these mice. Here, we show that the SV components of the EP electrogenic machinery and the epithelial barriers limiting the intrastrial fluid space, which are both necessary for the EP production, were preserved in Cx30−/− mice. In contrast, the endothelial barrier of the capillaries supplying the SV was disrupted before EP onset. This disruption is expected to result in an intrastrial electric shunt that is sufficient to account for the absence of the EP production. Immunofluorescence analysis of wild-type mice detected Cx30 in the basal and intermediate cells of the SV but not in the endothelial cells of the SV capillaries. Moreover, dye-coupling experiments showed that endothelial cells were not coupled to the SV basal, intermediate, and marginal cells. SV transcriptome analysis revealed a significant down-regulation of betaine homocysteine S-methyltransferase (Bhmt) in the Cx30−/− mice, which was restricted to the SV and resulted in a local increase in homocysteine, a known factor of endothelial dysfunction. Disruption of the SV endothelial barrier is a previously undescribed pathogenic process underlying hearing impairment.

Changes in MicroRNA Expression Contribute to Pancreatic β-Cell Dysfunction in Prediabetic NOD Mice

During the initial phases of type 1 diabetes, pancreatic islets are invaded by immune cells, exposing β-cells to proinflammatory cytokines. This unfavorable environment results in gene expression modifications leading to loss of β-cell functions. To study the contribution of microRNAs (miRNAs) in this process, we used microarray analysis to search for changes in miRNA expression in prediabetic NOD mice islets. We found that the levels of miR-29a/b/c increased in islets of NOD mice during the phases preceding diabetes manifestation and in isolated mouse and human islets exposed to proinflammatory cytokines. Overexpression of miR-29a/b/c in MIN6 and dissociated islet cells led to impairment in glucose-induced insulin secretion. Defective insulin release was associated with diminished expression of the transcription factor Onecut2, and a consequent rise of granuphilin, an inhibitor of β-cell exocytosis. Overexpression of miR-29a/b/c also promoted apoptosis by decreasing the level of the antiapoptotic protein Mcl1. Indeed, a decoy molecule selectively masking the miR-29 binding site on Mcl1 mRNA protected insulin-secreting cells from apoptosis triggered by miR-29 or cytokines. Taken together, our findings suggest that changes in the level of miR-29 family members contribute to cytokine-mediated β-cell dysfunction occurring during the initial phases of type 1 diabetes.

Targeting pannexin1 improves seizure outcome

Imbalance of the excitatory neurotransmitter glutamate and of the inhibitory neurotransmitter GABA is one of several causes of seizures. ATP has also been implicated in epilepsy. However, little is known about the mechanisms involved in the release of ATP from cells and the consequences of the altered ATP signaling during seizures. Pannexin1 (Panx1) is found in astrocytes and in neurons at high levels in the embryonic and young postnatal brain, declining in adulthood. Panx1 forms large-conductance voltage sensitive plasma membrane channels permeable to ATP that are also activated by elevated extracellular K+ and following P2 receptor stimulation. Based on these properties, we hypothesized that Panx1 channels may contribute to seizures by increasing the levels of extracellular ATP. Using pharmacological tools and two transgenic mice deficient for Panx1 we show here that interference with Panx1 ameliorates the outcome and shortens the duration of kainic acid-induced status epilepticus. These data thus indicate that the activation of Panx1 in juvenile mouse hippocampi contributes to neuronal hyperactivity in seizures.

Cx36 makes channels coupling human pancreatic β - cells, and correlates with insulin expression

Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observed in type 2 diabetes. Since human islets differ in several respects from those of laboratory rodents, we have now screened human pancreas, and islets isolated thereof, for expression of a variety of connexin genes, tested whether the cognate proteins form functional channels for islet cell exchanges, and assessed whether this expression changes with beta-cell function in islets of control and type 2 diabetics. Here, we show that (i) different connexin isoforms are differentially distributed in the exocrine and endocrine parts of the human pancreas; (ii) human islets express at the transcript level different connexin isoforms; (iii) the membrane of beta-cells harbors detectable levels of gap junctions made of Cx36; (iv) this protein is concentrated in lipid raft domains of the beta-cell membrane where it forms gap junctions; (v) Cx36 channels allow for the preferential exchange of cationic molecules between human beta-cells; (vi) the levels of Cx36 mRNA correlated with the expression of the insulin gene in the islets of both control and type 2 diabetics. The data show that Cx36 is a native protein of human pancreatic islets, which mediates the coupling of the insulin-producing beta-cells, and contributes to control beta-cell function by modulating gene expression.

Islet-cell-to-cell communication as basis for normal insulin secretion.

The emergence of pancreatic islets has necessitated the development of a signalling system for the intra‐ and inter‐islet coordination of β cells. With evolution, this system has evolved into a complex regulatory network of partially cross‐talking pathways, whereby individual cells sense the state of activity of their neighbours and, accordingly, regulate their own level of functioning. A consistent feature of this network in vertebrates is the expression of connexin (Cx)‐36‐made cell‐to‐cell channels, which cluster at gap junction domains of the cell membrane, and which adjacent β cells use to share cytoplasmic ions and small metabolites within individual islets. This chapter reviews what is known about Cx36, and the mechanism whereby this β‐cell connexin significantly regulates insulin secretion. It further outlines other less established functions of the protein and evaluates its potential relevance for the development of novel therapeutic approaches to diabetes.

Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (–/–) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence. In hetero- and homozygous (+/– and –/–) embryoid bodies, immunoblotting revealed a Triton-soluble, truncated form of cingulin, increased levels of the tight junction proteins ZO-2, occludin, claudin-6 and Lfc, and decreased levels of ZO-1. The +/– and –/– embryoid bodies contained epithelial cells with normal tight junctions, as determined by freeze-fracture and transmission electron microscopy, and a biotin permeability assay. The localization of ZO-1, occludin and claudin-6 appeared normal in mutant epithelial cells, indicating that cingulin is not required for their junctional recruitment. Real-time quantitative reverse-transcription PCR (real-time qRT-PCR) showed that differentiation of embryonic stem cells into embryoid bodies was associated with up-regulation of mRNAs for several tight junction proteins. Microarray analysis and real-time qRT-PCR showed that cingulin mutation caused a further increase in the transcript levels of occludin, claudin-2, claudin-6 and claudin-7, which were probably due to an increase in expression of GATA-6, GATA-4 and HNF-4α, transcription factors implicated in endodermal differentiation. Thus, lack of junctional cingulin does not prevent tight-junction formation, but gene expression and tight junction protein levels are altered by the cingulin mutation.

Connexins and secretion.

Summry— Connexin channels clustered at gap junctions are obligatory attributes of all macroscopic endocrine and exocrine glands investigated so far and also connect most types of cells which produce secretory products in other tissues. Increasing evidence indicates that connexins, and the cell‐to‐cell communications that these proteins permit, contribute to control the growth of secretory cells, their expression of specific genes and their differentiated function, including their characteristic ability to biosynthetize and release secretory products in a regulated manner. Since the previous reviews which have been published on this topic, several lines of evidence have been added in support of multiple regulatory roles of gland connexins. Here, we review this novel evidence, point to the many questions which are still open and discuss some interesting perspectives of the field.