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Philippe A Halban

Publications and source records attributed to Philippe A Halban.

At least 19 recordsLinked to original sources
PubMed2007

Pro-survival role of gelsolin in mouse beta-cells.

We have previously shown that the Ca(2+)-dependent actin-severing protein gelsolin plays an important role in regulated insulin secretion. The aim of this study was to determine the role of gelsolin in beta-cell survival as it has been shown to play a dual role in apoptosis in other cell types. MIN6 subclones B1 and C3, shown previously to express gelsolin at different levels (B1>>C3 cells), were used for this purpose. We demonstrate that B1 cells have lower levels of apoptosis and active caspase-3 when compared with C3 cells, in both standard (25 mmol/l glucose and 15% FCS) and deprived (5 mmol/l glucose and 1% FCS) conditions. Overexpression of gelsolin resulted in a decrease in the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)(+) and active caspase-3(+) cells. Conversely, knockdown of gelsolin by RNA interference in B1 cells caused an increase in the number of TUNEL(+) and active caspase-3(+) cells. Finally, the anti-apoptotic role of gelsolin was confirmed in purified primary mouse beta-cells where overexpression of gelsolin resulted in a decrease in the percentage of TUNEL(+) cells. In summary, our results show for the first time that gelsolin plays a pro-survival role in pancreatic beta-cells.

Animals↗
PubMed2006

Regulation of pancreatic beta-cell insulin secretion by actin cytoskeleton remodelling: role of gelsolin and cooperation with the MAPK signalling pathway.

We have previously isolated two MIN6 beta-cell sublines, B1, highly responsive to glucose-stimulated insulin secretion, and C3, markedly refractory (Lilla, V., Webb, G., Rickenbach, K., Maturana, A., Steiner, D. F., Halban, P. A. and Irminger, J. C. (2003) Endocrinology 144, 1368-1379). We now demonstrate that C3 cells have substantially increased amounts of F-actin stress fibres whereas B1 cells have shorter cortical F-actin. Consistent with these data, B1 cells display glucose-dependent actin remodelling whereas, in C3 cells, F-actin is refractory to this secretagogue. Furthermore, F-actin depolymerisation with latrunculin B restores glucose-stimulated insulin secretion in C3 cells. In parallel, glucose-stimulated ERK1/2 activation is greater in B1 than in C3 cells, and is potentiated in both sublines following F-actin depolymerisation. Glucose-activated phosphoERK1/2 accumulates at actin filament tips adjacent to the plasma membrane, indicating that these are the main sites of action for this kinase during insulin secretion. In addition, B1 cell expression of the calcium-dependent F-actin severing protein gelsolin is >100-fold higher than that of C3 cells. Knock-down of gelsolin reduced glucose-stimulated insulin secretion, whereas gelsolin over-expression potentiated secretion from B1 cells. Gelsolin localised along depolymerised actin fibres after glucose stimulation. Taken together, these data demonstrate that F-actin reorganization prior to insulin secretion requires gelsolin and plays a role in the glucose-dependent MAPK signal transduction that regulates beta-cell insulin secretion.

Actins↗
PubMed2006

Blockade of beta1 integrin-laminin-5 interaction affects spreading and insulin secretion of rat beta-cells attached on extracellular matrix.

When attached on a matrix produced by a rat bladder carcinoma cell line (804G matrix), rat pancreatic beta-cells spread in response to glucose and secrete more insulin compared with cells attached on poly-l-lysine. The aim of this study was to determine whether laminin-5 and its corresponding cell receptor beta1 integrin are implicated in these phenomena. By using specific blocking antibodies, we demonstrated that laminin-5 is the component present in 804G matrix responsible for the effect of 804G matrix on beta-cell function and spreading. When expression of two well-known laminin-5 ligands, beta1 and beta4 integrin, was assessed by Western blot and RT-PCR, only the beta1 integrin was detected in beta-cells. Anti-beta1 integrin antibody reduced the spreading of beta-cells on 804G matrix. Blockade of the interaction between beta1 integrins and laminin-5 resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti-beta1 integrin antibody also inhibited focal adhesion kinase phosphorylation induced by 804G matrix. In conclusion, anti-beta1 integrin and -laminin-5 antibodies interfere with spreading of beta-cells, resulting in decreased insulin secretion in response to glucose. Our findings indicate that outside-in signaling via engagement of beta1 integrins by laminin-5 is an important component of normal beta-cell function.

Animals↗
PubMed2006

Impaired NH2-terminal processing of human proislet amyloid polypeptide by the prohormone convertase PC2 leads to amyloid formation and cell death.

Islet amyloid, formed by aggregation of islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of the pancreas in type 2 diabetes and may contribute to the progressive loss of beta-cells in this disease. We tested the hypothesis that impaired processing of the IAPP precursor proIAPP contributes to amyloid formation and cell death. GH3 cells lacking the prohormone convertase 1/3 (PC1/3) and IAPP and with very low levels of prohormone convertase 2 (PC2) were transduced with adenovirus (Ad) expressing human or rat (control) proIAPP linked to green fluorescent protein, with or without Ad-PC2 or Ad-PC1/3. Expression of human proIAPP increased the number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells 96 h after transduction (+hIAPP 8.7 +/- 0.4% vs. control 3.0 +/- 0.4%; P < 0.05). COOH-terminal processing of human proIAPP by PC1/3 increased (hIAPP+PC1/3 10.4 +/- 0.7%; P < 0.05), whereas NH(2)-terminal processing of proIAPP by addition of PC2 markedly decreased (hIAPP+PC2 5.5 +/- 0.5%; P < 0.05) the number of apoptotic GH3 cells. Islets from mice lacking PC2 and with beta-cell expression of human proIAPP (hIAPP(+/+)/PC2(-/-)) developed amyloid associated with beta-cell death during 2-week culture. Rescue of PC2 expression by ex vivo transduction with Ad-PC2 restored NH(2)-terminal processing to mature IAPP and decreased both the extent of amyloid formation and the number of TUNEL-positive cells (-PC2 26.5 +/- 4.1% vs. +PC2 16.1 +/- 4.3%; P < 0.05). These findings suggest that impaired NH(2)-terminal processing of proIAPP leads to amyloid formation and cell death and that accumulation of the NH(2)-terminally extended human proIAPP intermediate may be a critical initiating step in amyloid formation.

Adenoviridae↗
PubMed2005

Activation of NF-kappaB by extracellular matrix is involved in spreading and glucose-stimulated insulin secretion of pancreatic beta cells.

Laminin-5-rich extracellular matrix derived from 804G cells (804G-ECM) engages beta1 integrins to induce spreading, improve glucose-stimulated insulin secretion (GSIS), and increase survival of pancreatic beta cells. The present study examines whether 804G-ECM activates the transcriptional activity of NF-kappaB and the involvement of NF-kappaB in those effects of 804G-ECM on pancreatic beta cells. 804G-ECM induces nuclear translocation and the DNA binding activity of the p65 subunit of NF-kappaB. 804G-ECM-induced nuclear translocation of NF-kappaB was weak as compared with that induced by interleukin-1beta. Transient 804G-ECM-induced DNA binding activity of NF-kappaB (peak at 2 h) and overexpression of NF-kappaB target genes IkappaB alpha and NF-kappaB1(p105) (peak at 4 h) were observed. When NF-kappaB was inhibited by an inhibitor of IkappaB alpha phosphorylation (Bay 11-7082) or by a recombinant adenovirus expressing the nonphosphorylatable form of IkappaB alpha, 804G-ECM-induced cell spreading and actin cytoskeleton organization were reduced. GSIS from cells on 804G-ECM was inhibited 5-fold, whereas cell survival was not affected. In summary, the results indicate that 804G-ECM induces a transient and moderate NF-kappaB activity. This study shows for the first time that ECM-induced NF-kappaB activity is necessary in maintaining GSIS, although it does not affect survival of pancreatic beta cells. The effects of ECM-induced NF-kappaB activity contrast with the deleterious effects of cytokine-induced NF-kappaB activity. It is proposed that transient and moderate NF-kappaB activity is essential for proper function of the pancreatic beta cell.

Actins↗
PubMed2004

Leptin modulates beta cell expression of IL-1 receptor antagonist and release of IL-1beta in human islets.

High concentrations of glucose induce beta cell production of IL-1beta, leading to impaired beta cell function and apoptosis in human pancreatic islets. IL-1 receptor antagonist (IL-1Ra) is a naturally occurring antagonist of IL-1beta and protects cultured human islets from glucotoxicity. Therefore, the balance of IL-1beta and IL-1Ra may play a crucial role in the pathogenesis of diabetes. In the present study, we observed expression of IL-1Ra in human pancreatic beta cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro, chronic exposure of human islets to leptin, a hormone secreted by adipocytes, decreased beta cell production of IL-1Ra and induced IL-1beta release from the islet preparation, leading to impaired beta cell function, caspase-3 activation, and apoptosis. Exogenous addition of IL-1Ra protected cultured human islets from the deleterious effects of leptin. Antagonizing IL-1Ra by introduction of small interfering RNA to IL-1Ra into human islets led to caspase-3 activation, DNA fragmentation, and impaired beta cell function. Moreover, siIL-1Ra enhanced glucose-induced beta cell apoptosis. These findings demonstrate expression of IL-1Ra in the human beta cell, providing localized protection against leptin- and glucose-induced islet IL-1beta.

Apoptosis↗
PubMed2004

Cellular sources of new pancreatic beta cells and therapeutic implications for regenerative medicine.

Replacing missing insulin-producing beta cells to treat diabetes is a major challenge for regenerative medicine. A better understanding of beta-cell embryogenesis and regeneration in adult life is needed to devise means to derive these specialized cells in sufficiently large numbers from stem or precursor cells. It is also critical to ensure that any surrogate or regenerated beta cells have perfectly regulated insulin production, which is essential for physiological glucose homeostasis.

Adult↗
PubMed2004

Sorting ourselves out: seeking consensus on trafficking in the beta-cell.

Biogenesis of the regulated secretory pathway in the pancreatic beta-cell involves packaging of products, notably proinsulin, into immature secretory granules derived from the trans-Golgi network. Proinsulin is converted to insulin and C-peptide as granules mature. Secretory proteins not entering granules are conveyed by transport intermediates directly to the plasma membrane for constitutive secretion. One of the co-authors, Peter Arvan, has proposed that in addition, small vesicles bud from granules to traffic to the endosomal system. From there, some proteins are secreted by a (post-granular) constitutive-like pathway. He argues that retention in granules is facilitated by condensation, rendering soluble products (notably C-peptide and proinsulin) more available for constitutive-like secretion. Thus he argues that prohormone conversion is potentially important in secretory granule biogenesis. The other co-author, Philippe Halban, argues that the post-granular secretory pathway is not of physiological relevance in primary beta-cells, and contests the importance of proinsulin conversion for retention in granules. Both, however, agree that trafficking from granules to endosomes is important, purging granules of unwanted newly synthesized proteins and allowing their traffic to other destinations. In this Traffic Interchange, the two co-authors attempt to reconcile their differences, leading to a common vision of proinsulin trafficking in primary and transformed cells.

Animals↗
PubMed2004

Role of beta-cell prohormone convertase (PC)1/3 in processing of pro-islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) (amylin), the major component of islet amyloid, is produced by cleavage at the COOH- and NH(2)-termini of its precursor, proIAPP, likely by the beta-cell prohormone convertases (PC) 1/3 and PC2. Mice lacking PC2 can process proIAPP at its COOH- but not its NH(2)-terminal cleavage site, suggesting that PC1/3 is capable of initiating proIAPP cleavage at its COOH-terminus. To determine the precise role of PC1/3 in proIAPP processing, Western blot analysis was performed on islets isolated from mice lacking PC1/3 (PC1/3(-/-)). These islets contained not only fully processed IAPP as in PC1/3(+/+) islets, but also elevated levels of a COOH-terminally unprocessed intermediate form, suggesting impaired processing at the COOH-terminus. Next, GH3 cells that do not normally express proIAPP or detectable levels of PC1/3 or PC2 were cotransduced with adenoviruses expressing rat proIAPP and either PC2 or PC1/3. As expected, in GH3 cells transduced to express only proIAPP, no processing was observed. Coexpression of proIAPP and PC2 resulted in production of mature IAPP, whereas in cells that coexpressed proIAPP and PC1/3 only a 6-kDa intermediate was produced. We conclude that PC1/3 is important for processing of proIAPP at the COOH-terminus, but in its absence, PC2 can initiate complete processing of proIAPP to IAPP by cleaving the precursor at either its NH(2)- or COOH-terminal cleavage sites.

Amyloid↗
PubMed2004

Extracellular matrix protects pancreatic beta-cells against apoptosis: role of short- and long-term signaling pathways.

We have shown previously that culture of beta-cells on matrix derived from 804G cells and rich in laminin-5 improves their function. The purpose of this study was to investigate whether this matrix protects beta-cells against apoptosis and to elucidate signaling pathways involved. Matrix protected sorted rat beta-cells against apoptosis under standard conditions (11.2 mmol/l glucose, 10% serum), after serum deprivation (1% serum), and in response to interleukin-1beta (IL-1beta; 2 ng/ml), compared with control (poly-L-lysine [pLL]). Caspase-8 activity was reduced in cells cultured on matrix, whereas focal adhesion kinase (FAK), protein kinase B (PKB, or Akt), and extracellular signal-regulated kinase (ERK) phosphorylation was augmented. Treatment (4 h) with an anti-beta1 integrin antibody, with the ERK pathway inhibitor PD98059, and/or with the phosphatidylinositol 3-kinase inhibitor LY294002 augmented cell death on 804G matrix but not on pLL. In long-term assays (48 h), PD98059 but not LY294002 drastically augmented cell death on 804G matrix but did so to a lesser extent on pLL. The protein inhibitor of nuclear factor-kappaB (IkappaBalpha) was overexpressed in cells cultured 18 h on matrix with partial blockade by PD98059. In summary, this study provides evidence for activation of signaling pathways and gene expression by extracellular matrix leading to improved beta-cell survival.

Animals↗
PubMed2003

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex.

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.

Actins↗
PubMed2003

Mutant proinsulin that cannot be converted is secreted efficiently from primary rat beta-cells via the regulated pathway.

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called "constitutive-like" pathway. To address this directly, mutant human proinsulin (Arg/Gly(32):Lys/Thr(64)), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin.

Acetylcysteine↗
PubMed2003

Differential gene expression in well-regulated and dysregulated pancreatic beta-cell (MIN6) sublines.

To identify genes involved in regulated insulin secretion, we have established and characterized two sublines derived from the mouse pancreatic beta-cell line MIN6, designated B1 and C3. They have a similar insulin content, but differ in their secretory properties. B1 responded to glucose in a concentration- and cell confluence-dependent manner, whereas C3 did not. B1 cells were stimulated by phorbol 12-myristate 13-acetate, leucine, arginine, glibenclamide, isobutylmethylxanthine, and KCl, whereas C3 did not respond (leucine, arginine, and glibenclamide) or responded to a lesser extent (isobutylmethylxanthine, phorbol 12-myristate 13-acetate, and KCl). Although intracellular Ca(2+) rose in response to glucose in B1 but not C3 cells, KCl increased intracellular Ca(2+) in a similar manner in both sublines. GLUT-1, GLUT-2, Kir6.2, and SUR1 expression was not significantly different between B1 and C3 cells, whereas E-cadherin was more abundantly expressed in B1 cells. A more complete list of differentially expressed genes was established by suppression subtractive hybridization and high density (Affymetrix) oligonucleotide microarrays. Genes were clustered according to known or putative function. Those involved in metabolism, intracellular signaling, cytoarchitecture, and cell adhesion are of potential interest. These two sublines should be useful for identification of the genes and mechanisms involved in regulated insulin secretion of the pancreatic beta-cell.

Animals↗
PubMed2003

Dominant negative pathogenesis by mutant proinsulin in the Akita diabetic mouse.

Autosomal dominant diabetes in the Akita mouse is caused by mutation of the insulin 2 gene, whose product replaces a cysteine residue that is engaged in the formation of an intramolecular disulfide bond. These heterozygous mice exhibit severe insulin deficiency despite coexpression of normal insulin molecules derived from three other wild-type alleles of the insulin 1 and 2 genes. Although the results of our previous study suggested that the mutant proinsulin 2 is misfolded and blocked in the transport from the endoplasmic reticulum to the Golgi apparatus, its dominant negative nature has not been fully characterized. In the present study, we investigated the possible pathogenic mechanisms induced by the mutant proinsulin 2. There is no evidence that the mutant proinsulin 2 attenuates the overall protein synthesis rate or promotes the formation of aberrant disulfide bonds. The trafficking of constitutively secreted alkaline phosphatase, however, is significantly decreased in the islets of Akita mice, indicating that the function of early secretory pathways is nonspecifically impaired. Morphological analysis has revealed that secretory pathway organelle architecture is progressively devastated in the beta-cells of Akita mice. These findings suggest that the organelle dysfunction resulting from the intracellular accumulation of misfolded proinsulin 2 is primarily responsible for the defect of coexisting wild-type insulin secretion in Akita beta-cells.

Amino Acid Substitution↗
PubMed2002

Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis.

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and augments beta cell mass via activation of beta cell proliferation and islet neogenesis. We examined whether GLP-1 receptor signaling modifies the cellular susceptibility to apoptosis. Mice administered streptozotocin (STZ), an agent known to induce beta cell apoptosis, exhibit sustained improvement in glycemic control and increased levels of plasma insulin with concomitant administration of the GLP-1 agonist exendin-4 (Ex-4). Blood glucose remained significantly lower for weeks after cessation of exendin-4. STZ induced beta cell apoptosis, which was significantly reduced by co-administration of Ex-4. Conversely, mice with a targeted disruption of the GLP-1 receptor gene exhibited increased beta cell apoptosis after STZ administration. Exendin-4 directly reduced cytokine-induced apoptosis in purified rat beta cells exposed to interleukin 1beta, tumor necrosis fator alpha, and interferon gamma in vitro. Furthermore, Ex-4-treated BHK-GLP-1R cells exhibited significantly increased cell viability, reduced caspase activity, and decreased cleavage of beta-catenin after treatment with cycloheximide in vitro. These findings demonstrate that GLP-1 receptor signaling directly modifies the susceptibility to apoptotic injury, and provides a new potential mechanism linking GLP-1 receptor activation to preservation or enhancement of beta cell mass in vivo.

Animals↗
PubMed2002

FLIP switches Fas-mediated glucose signaling in human pancreatic beta cells from apoptosis to cell replication.

Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic beta cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of beta cell turnover. In human islets, elevated glucose concentrations impair beta cell proliferation and induce beta cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic beta cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive beta cells; FLIP was no longer detectable in such TUNEL-positive beta cells. Up-regulation of FLIP, by incubation with transforming growth factor beta or by transfection with an expression vector coding for FLIP, protected beta cells from glucose-induced apoptosis, restored beta cell proliferation, and improved beta cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human beta cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.

Aged↗