Christophe Broca

GLP-1 Mediates Antiapoptotic Effect by Phosphorylating Bad through a β-Arrestin 1-mediated ERK1/2 Activation in Pancreatic β-Cells

Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the β-cells is of major importance. In β-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Gαs/cAMP/cAMP-dependent protein kinase (PKA) or β-arrestin 1, a scaffold protein. Using pharmacological inhibitors, β-arrestin 1 small interfering RNA, and islets isolated from β-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the β-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the β-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the β-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. β-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in β-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a β-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of β-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.

ERK1/2 Control Phosphorylation and Protein Level of cAMP-Responsive Element–Binding Protein: A Key Role in Glucose-Mediated Pancreatic β-Cell Survival

cAMP-responsive element–binding protein (CREB) is required for β-cell survival by regulating expression of crucial genes such as bcl-2 and IRS-2. Using MIN6 cells and isolated rat pancreatic islets, we investigated the signaling pathway that controls phosphorylation and protein level of CREB. We observed that 10 mmol/l glucose–induced CREB phosphorylation was totally inhibited by the protein kinase A (PKA) inhibitor H89 (2 μmol/l) and reduced by 50% with the extracellular signal–regulated kinase (ERK)1/2 inhibitor PD98059 (20 μmol/l). This indicates that ERK1/2, reported to be located downstream of PKA, participates in the PKA-mediated CREB phosphorylation elicited by glucose. In ERK1/2-downregulated MIN6 cells by siRNA, glucose-stimulated CREB phosphorylation was highly reduced and CREB protein content was decreased by 60%. In MIN6 cells and islets cultured for 24–48 h in optimal glucose concentration (10 mmol/l), which promotes survival, blockade of ERK1/2 activity with PD98059 caused a significant decrease in CREB protein level, whereas CREB mRNA remained unaffected (measured by real-time quantitative PCR). This was associated with loss of bcl-2 mRNA and protein contents, caspase-3 activation, and emergence of ultrastructural apoptotic features detected by electron microscopy. Our results indicate that ERK1 and -2 control the phosphorylation and protein level of CREB and play a key role in glucose-mediated pancreatic β-cell survival.

Degradation of cAMP-Responsive Element–Binding Protein by the Ubiquitin-Proteasome Pathway Contributes to Glucotoxicity in β-Cells and Human Pancreatic Islets

OBJECTIVE In type 2 diabetes, chronic hyperglycemia is detrimental to β-cells, causing apoptosis and impaired insulin secretion. The transcription factor cAMP-responsive element–binding protein (CREB) is crucial for β-cell survival and function. We investigated whether prolonged exposure of β-cells to high glucose affects the functional integrity of CREB. RESEARCH DESIGN AND METHODS INS-1E cells and rat and human islets were used. Gene expression was analyzed by RT-PCR and Western blotting. Apoptosis was detected by cleaved caspase-3 emergence, DNA fragmentation, and electron microscopy. RESULTS Chronic exposure of INS-1E cells and rat and human islets to high glucose resulted in decreased CREB protein expression, phosphorylation, and transcriptional activity associated with apoptosis and impaired β-cell function. High-glucose treatment increased CREB polyubiquitination, while treatment of INS-1E cells with the proteasome inhibitor MG-132 prevented the decrease in CREB content. The emergence of apoptosis in INS-1E cells with decreased CREB protein expression knocked down by small interfering RNA suggested that loss of CREB protein content induced by high glucose contributes to β-cell apoptosis. Loading INS-1E cells or human islets with a cell-permeable peptide mimicking the proteasomal targeting sequence of CREB blocked CREB degradation and protected INS-1E cells and human islets from apoptosis induced by high glucose. The insulin secretion in response to glucose and the insulin content were preserved in human islets exposed to high glucose and loaded with the peptide. CONCLUSIONS These studies demonstrate that the CREB degradation by the ubiquitin-proteasome pathway contributes to β-cell dysfunction and death upon glucotoxicity and provide new insight into the cellular mechanisms of glucotoxicity.

β-Arrestin 1 Is Required for PAC1 Receptor-mediated Potentiation of Long-lasting ERK1/2 Activation by Glucose in Pancreatic β-Cells

In pancreatic β-cells, the pituitary adenylate cyclase-activating polypeptide (PACAP) exerts a potent insulin secretory effect via PAC1 and VPAC receptors (Rs) through the Gαs/cAMP/protein kinase A pathway. Here, we investigated the mechanisms linking PAC1R to ERK1/2 activation in INS-1E β-cells and pancreatic islets. PACAP caused a transient (5 min) increase in ERK1/2 phosphorylation via PAC1Rs and promoted nuclear translocation of a fraction of cytosolic p-ERK1/2. Both protein kinase A- and Src-dependent pathways mediated this transient ERK1/2 activation. Moreover, PACAP potentiated glucose-induced long-lasting ERK1/2 activation. Blocking Ca2+ influx abolished glucose-induced ERK1/2 activation and PACAP potentiating effect. Glucose stimulation during KCl depolarization showed that, in addition to the triggering signal (rise in cytosolic [Ca2+]), the amplifying pathway was also involved in glucose-induced sustained ERK1/2 activation and was required for PACAP potentiation. The finding that at 30 min glucose-induced p-ERK1/2 was detected in both cytosol and nucleus while the potentiating effect of PACAP was only observed in the cytosol, suggested the involvement of the scaffold protein β-arrestin. Indeed, β-arrestin 1 (β-arr1) depletion (in β-arr1 knockout mouse islets or in INS-1E cells by siRNA) completely abolished PACAP potentiation of long-lasting ERK1/2 activation by glucose. Finally, PACAP potentiated glucose-induced CREB transcriptional activity and IRS-2 mRNA expression mainly via the ERK1/2 signaling pathway, and likewise, β-arr1 depletion reduced the PACAP potentiating effect on IRS-2 expression. These results establish for the first time that PACAP potentiates glucose-induced long-lasting ERK1/2 activation via a β-arr1-dependent pathway and thus provide new insights concerning the mechanisms of PACAP and glucose actions in pancreatic β-cells.

Acute nutrient regulation of the mitochondrial glutathione redox state in pancreatic β-cells

The glucose stimulation of insulin secretion by pancreatic β-cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in β-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of EGSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in β-cells and reacted to low concentrations of exogenous H2O2 and menadione. Under control conditions, cytosolic/nuclear EGSH was close to -300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial EGSH was less negative and rapidly regulated by glucose and other nutrients, ranging from -280 mV in the absence of glucose to -299 mV in 30 mM glucose. These changes were largely independent from changes in intracellular Ca(2+) concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial EGSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, EGSH in pancreatic β-cells under control conditions.

Calcitonin gene-related peptide immunoreactivity in the rat efferent vestibular system during development

The organization of the efferent fiber network during postnatal development was investigated by immunocytochemical detection of the calcitonin gene-related peptide (CGRP) in rat vestibular receptors from postnatal day 0 (PD 0) to adulthood. CGRP was detected at birth in a few efferent fibers below the sensory epithelia of cristae and maculae. Thereafter, the nerve fibers in the cristae progressively invaded the epithelia with an apex to base gradient from PD 2 to PD 4. There was also a rearrangement of the fibers during maturation of the efferent innervation, such that after reaching the surface of the epithelium, they turned back and moved towards the base of the sensory cells, producing numerous synaptic contacts. Analysis of surface preparations of utricules showed the irregular and asymmetric topographic organization of the efferent fiber network and the extensive, complex distribution of this innervation. The presence and broad distribution of CGRP in the epithelium at critical stages of development and synaptogenesis suggests that it is involved in the maturation of vestibular receptors.

Proteasome Dysfunction Mediates High Glucose-Induced Apoptosis in Rodent Beta Cells and Human Islets

The ubiquitin/proteasome system (UPS), a major cellular protein degradation machinery, plays key roles in the regulation of many cell functions. Glucotoxicity mediated by chronic hyperglycaemia is detrimental to the function and survival of pancreatic beta cells. The aim of our study was to determine whether proteasome dysfunction could be involved in beta cell apoptosis in glucotoxic conditions, and to evaluate whether such a dysfunction might be pharmacologically corrected. Therefore, UPS activity was measured in GK rats islets, INS-1E beta cells or human islets after high glucose and/or UPS inhibitor exposure. Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. In vitro, we found that chronic exposure of INS-1E cells to high glucose concentrations significantly decreases the three proteasome activities by 20% and leads to caspase-3-dependent apoptosis. We showed that pharmacological blockade of UPS activity by 20% leads to apoptosis in a same way. Indeed, ER stress was involved in both conditions. These results were confirmed in human islets, and proteasome activities were also decreased in hyperglycemic GK rats islets. Moreover, we observed that a high glucose treatment hypersensitized beta cells to the apoptotic effect of proteasome inhibitors. Noteworthily, the decreased proteasome activity can be corrected with Exendin-4, which also protected against glucotoxicity-induced apoptosis. Taken together, our findings reveal an important role of proteasome activity in high glucose-induced beta cell apoptosis, potentially linking ER stress and glucotoxicity. These proteasome dysfunctions can be reversed by a GLP-1 analog. Thus, UPS may be a potent target to treat deleterious metabolic conditions leading to type 2 diabetes.

Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4.

Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells.

Proteasomal degradation of the histone acetyl transferase p300 contributes to beta-cell injury in a diabetes environment

In type 2 diabetes, amyloid oligomers, chronic hyperglycemia, lipotoxicity, and pro-inflammatory cytokines are detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The histone acetyl transferase p300, involved in remodeling of chromatin structure by epigenetic mechanisms, is a key ubiquitous activator of the transcriptional machinery. In this study, we report that loss of p300 acetyl transferase activity and expression leads to beta-cell apoptosis, and most importantly, that stress situations known to be associated with diabetes alter p300 levels and functional integrity. We found that proteasomal degradation is the mechanism subserving p300 loss in beta-cells exposed to hyperglycemia or pro-inflammatory cytokines. We also report that melatonin, a hormone produced in the pineal gland and known to play key roles in beta-cell health, preserves p300 levels altered by these toxic conditions. Collectively, these data imply an important role for p300 in the pathophysiology of diabetes.

CO-72: L'inhibition de la kinase Tpl2 protège contre l'apoptose et la dysfonction des cellules bêta pancréatiques en conditions pro-inflammatoires : une nouvelle stratégie antidiabétique potentielle ?

Introduction Dans les contextes du diabete de type 1, de type 2, et de la greffe dilots de Langerhans, les molecules cles pour la mediation de linflammation au sein des cellules beta sont des cibles majeures pour des strategies therapeutiques innovantes. Nous avons teste lhypothese selon laquelle la MAP3 Kinase 8 Tpl2 (Tumor progression locus 2) pouvait etre un acteur important dans le reseau de signalisation implique dans les effets deleteres de plusieurs cytokines pro-inflammatoires sur la fonction et la survie des cellules beta. Materiels et Methodes La fonction et la survie des cellules beta INS-1E, des ilots pancreatiques murins et humains sont mesurees par activation de cas-pase-3/fragmentation de lADN, et la secretion dinsuline en incubations statiques. Les niveaux dexpression/phosphorylation des proteines sont mesures par western blot. La tolerance au glucose, ainsi que les taux plas-matiques de glucose et dinsuline sont mesures chez les souris db/db. Resultats Nous montrons que la kinase Tpl2 est exprimee dans les cellules INS-1E et les ilots pancreatiques murins et humains. Tpl2 est activee et surexprimee par lIL-1beta et lassociation de plusieurs cytokines (IL-1beta + TNFalpha + IFNgamma). Son inhibition ne modifie pas les effets benefiques du glucose sur la survie des cellules beta, la secretion dinsuline, et lactivation des ERK1/2, mais bloque lactivation des MAP kinases p38, JNK, et ERK1/2 induite par les cytokines. Linhibition de Tpl2 reduit les effets pro-apoptotiques de lIL-1beta et de lassociation de cytokines pro-inflammatoires. La secretion dinsuline et la survie des ilots murins et humains traites avec linhibiteur pharmacologique de Tpl2 sont protegees des effets deleteres de linflammation. In vivo, ladministration de linhibiteur de Tpl2 a des souris diabetiques db/db ameliore la tolerance au glucose, reduit lhyperglycemie et lhyperinsulinemie. Conclusions Cette etude identifie Tpl2 comme un nouvel acteur moleculaire majeur controlant specifiquement les effets deleteres des cytokines pro-inflammatoires, et propose Tpl2 comme cible therapeutique visant a proteger les cellules beta de la dysfunction et destruction induites par linflammation.