10 results on '"Stiernet, Patrick"'
Search Results
2. In Vivo and In Vitro Glucose-Induced Biphasic Insulin Secretion in the Mouse: Pattern and Role of Cytoplasmic Ca2+ and Amplification Signals in β-Cells
- Author
-
Henquin, Jean-Claude, Nenquin, Myriam, Stiernet, Patrick, and Ahren, Bo
- Published
- 2006
3. Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum, are differentially regulated by phosphorylation of their penultimate threonine.
- Author
-
UCL - SSS/IREC - Institut de recherche expérimentale et clinique, UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, UCL - SST/ISV - Institut des sciences de la vie, Bobik, Krzysztof, Duby, Geoffrey, Nizet, Yannick, Vandermeeren, Caroline, Stiernet, Patrick, Kanczewska, Justyna, Boutry, Marc, UCL - SSS/IREC - Institut de recherche expérimentale et clinique, UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, UCL - SST/ISV - Institut des sciences de la vie, Bobik, Krzysztof, Duby, Geoffrey, Nizet, Yannick, Vandermeeren, Caroline, Stiernet, Patrick, Kanczewska, Justyna, and Boutry, Marc
- Abstract
Summary PMA2 and PMA4 are the most widely expressed plasma membrane H(+)-ATPases in Nicotiana plumbaginifolia and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised N. tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, allowing their individual purification. Using these tools, we showed that phosphorylation of the penultimate Thr of both PMA was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than one-third of PMA2 or PMA4 was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, further support for these two H(+)-ATPase subfamilies having different properties.
- Published
- 2010
4. Glucose-induced cytosolic pH changes in beta-cells and insulin secretion are not causally related: studies in islets lacking the Na+/H+ exchangeR NHE1.
- Author
-
UCL - MD/FSIO - Département de physiologie et pharmacologie, Stiernet, Patrick, Nenquin, Myriam, Moulin, Pierre, Jonas, Jean-Christophe, Henquin, Jean-Claude, UCL - MD/FSIO - Département de physiologie et pharmacologie, Stiernet, Patrick, Nenquin, Myriam, Moulin, Pierre, Jonas, Jean-Christophe, and Henquin, Jean-Claude
- Abstract
The contribution of Na(+)/H(+) exchange (achieved by NHE proteins) to the regulation of beta-cell cytosolic pH(c), and the role of pH(c) changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca(2+) ([Ca(2+)](c)) and pH(c) in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO(-)(3)/CO(2) or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO(-)(3)-dependent mechanisms in the correction of an acidification imposed by a pulse of NH(4)Cl. In contrast, basal pH(c) (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na(+)/H(+) exchange, did not affect pH(c) but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca(2+)](c) and insulin secretion in HCO(-)(3) and HEPES buffer, although pH(c) changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca(2+)](c) is clamped at an elevated level by KCl was also unrelated to pH(c) and pH(c) changes. All effects of glucose on [Ca(2+)](c) and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects beta-cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH(c) produced by glucose involve HCO(-)(3)-dependent mechanisms. Variations in beta-cell pH(c) are not causally related to changes in insulin secretion.
- Published
- 2007
5. Glucose acutely decreases pH of secretory granules in mouse pancreatic islets. Mechanisms and influence on insulin secretion
- Author
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UCL - MD/FSIO - Département de physiologie et pharmacologie, UCL - MD/MNOP - Département de morphologie normale et pathologique, UCL - (SLuc) Service d'anatomie pathologique, UCL - (SLuc) Autre, Stiernet, Patrick, Guiot, Yves, Gilon, Patrick, Henquin, Jean-Claude, UCL - MD/FSIO - Département de physiologie et pharmacologie, UCL - MD/MNOP - Département de morphologie normale et pathologique, UCL - (SLuc) Service d'anatomie pathologique, UCL - (SLuc) Autre, Stiernet, Patrick, Guiot, Yves, Gilon, Patrick, and Henquin, Jean-Claude
- Abstract
Glucose-induced insulin secretion requires a rise in beta-cell cytosolic Ca2+ ([Ca2+]c) that triggers exocytosis and a mechanistically unexplained amplification of the action of [Ca2+]c. Insulin granules are kept acidic by luminal pumping of protons with simultaneous Cl- uptake to maintain electroneutrality. Experiments using patched, dialyzed beta-cells prompted the suggestion that acute granule acidification by glucose underlies amplification of insulin secretion. However, others found glucose to increase granular pH in intact islets. In this study, we measured islet granular pH with Lysosensor DND-160, a fluorescent dye that permits ratiometric determination of pH < 6 in acidic compartments. Stimulation of mouse islets with glucose reversibly decreased granular pH by mechanisms that are dependent on metabolism and Cl- ions but independent of changes in [Ca2+]c and protein kinase A or C activity. Granular pH was increased by concanamycin (blocker of the vesicular type H+-ATPase) > methylamine (weak base) > Cl- omission. Concanamycin and methylamine did not alter glucose-induced [Ca2+]c increase in islets but strongly inhibited the two phases of insulin secretion. Omission of Cl- did not affect the first phase but decreased the second phase of both [Ca2+]c and insulin responses. Neither experimental condition affected the [Ca2+]c rise induced by 30 mM KCl, but the insulin responses were inhibited by concanamycin > methylamine and not affected by Cl- omission. The amplification of insulin secretion by glucose was not suppressed. We conclude that an acidic granular pH is important for insulin secretion but that the acute further acidification produced by glucose is not essential for the augmentation of secretion via the amplifying pathway.
- Published
- 2006
6. Glucose-induced Cytosolic pH Changes in β-Cells and Insulin Secretion Are Not Causally Related
- Author
-
Stiernet, Patrick, primary, Nenquin, Myriam, additional, Moulin, Pierre, additional, Jonas, Jean-Christophe, additional, and Henquin, Jean-Claude, additional
- Published
- 2007
- Full Text
- View/download PDF
7. Glucose Acutely Decreases pH of Secretory Granules in Mouse Pancreatic Islets
- Author
-
Stiernet, Patrick, primary, Guiot, Yves, additional, Gilon, Patrick, additional, and Henquin, Jean-Claude, additional
- Published
- 2006
- Full Text
- View/download PDF
8. Two widely expressed plasma membrane H+-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.
- Author
-
Bobik, Krzysztof, Duby, Geoffrey, Nizet, Yannick, Vandermeeren, Caroline, Stiernet, Patrick, Kanczewska, Justyna, and Boutry, Marc
- Subjects
CELL membranes ,NICOTIANA ,PHOSPHORYLATION ,SACCHAROMYCES cerevisiae ,TRANSGENIC plants - Abstract
The plasma membrane H
+ -ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H+ -ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H+ -ATPase subfamilies having different properties. [ABSTRACT FROM AUTHOR]- Published
- 2010
- Full Text
- View/download PDF
9. In vivo and in vitro glucose-induced biphasic insulin secretion in the mouse: pattern and role of cytoplasmic Ca2+ and amplification signals in beta-cells.
- Author
-
Henquin J, Nenquin M, Stiernet P, Ahren B, Henquin, Jean-Claude, Nenquin, Myriam, Stiernet, Patrick, and Ahren, Bo
- Abstract
The mechanisms underlying biphasic insulin secretion have not been completely elucidated. We compared the pattern of plasma insulin changes during hyperglycemic clamps in mice to that of glucose-induced insulin secretion and cytosolic calcium concentration ([Ca(2+)](c)) changes in perifused mouse islets. Anesthetized mice were infused with glucose to clamp blood glucose at 8.5 (baseline), 11.1, 16.7, or 30 mmol/l. A first-phase insulin response consistently peaked at 1 min, and a slowly ascending second phase occurred at 16.7 and 30 mmol/l glucose. Glucose-induced insulin secretion in vivo is thus biphasic, with a similarly increasing second phase in the mouse as in humans. In vitro, square-wave stimulation from a baseline of 3 mmol/l glucose induced similar biphasic insulin secretion and [Ca(2+)](c) increases, with sustained and flat second phases. The glucose dependency (3-30 mmol/l) of both changes was sigmoidal with, however, a shift to the right of the relation for insulin secretion compared with that for [Ca(2+)](c). The maximum [Ca(2+)](c) increase was achieved by glucose concentrations, causing half-maximum insulin secretion. Because this was true for both phases, we propose that contrary to current concepts, amplifying signals are also implicated in first-phase glucose-induced insulin secretion. To mimic in vivo conditions, islets were stimulated with high glucose after being initially perifused with 8.5 instead of 3.0 mmol/l glucose. First-phase insulin secretion induced by glucose at 11.1, 16.7, and 30 mmol/l was decreased by approximately 50%, an inhibition that could not be explained by commensurate decreases in [Ca(2+)](c) or in the pool of readily releasable granules. Also unexpected was the gradually ascending pattern of the second phase, now similar to that in vivo. These observations indicated that variations in prestimulatory glucose can secondarily affect the magnitude and pattern of subsequent glucose-induced insulin secretion. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
10. Glucose-induced cytosolic pH changes in beta-cells and insulin secretion are not causally related: studies in islets lacking the Na+/H+ exchangeR NHE1.
- Author
-
Stiernet P, Nenquin M, Moulin P, Jonas JC, and Henquin JC
- Subjects
- Animals, Biological Transport, Cation Transport Proteins, Dose-Response Relationship, Drug, Hydrogen-Ion Concentration, Insulin Secretion, Membrane Proteins, Mice, Mice, Inbred C57BL, Models, Biological, Protein Isoforms, Sodium-Hydrogen Exchanger 1, Sodium-Hydrogen Exchangers metabolism, Time Factors, Cytosol metabolism, Glucose metabolism, Insulin metabolism, Insulin-Secreting Cells metabolism
- Abstract
The contribution of Na(+)/H(+) exchange (achieved by NHE proteins) to the regulation of beta-cell cytosolic pH(c), and the role of pH(c) changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca(2+) ([Ca(2+)](c)) and pH(c) in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO(-)(3)/CO(2) or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO(-)(3)-dependent mechanisms in the correction of an acidification imposed by a pulse of NH(4)Cl. In contrast, basal pH(c) (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na(+)/H(+) exchange, did not affect pH(c) but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca(2+)](c) and insulin secretion in HCO(-)(3) and HEPES buffer, although pH(c) changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca(2+)](c) is clamped at an elevated level by KCl was also unrelated to pH(c) and pH(c) changes. All effects of glucose on [Ca(2+)](c) and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects beta-cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH(c) produced by glucose involve HCO(-)(3)-dependent mechanisms. Variations in beta-cell pH(c) are not causally related to changes in insulin secretion.
- Published
- 2007
- Full Text
- View/download PDF
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