scholarly journals Inositol Hexakisphosphate Primes Syndapin I/PACSIN 1 Activation In Endocytosis

2021 ◽  
Author(s):  
Yue Shi ◽  
Kaixuan Zhao ◽  
Guang Yang ◽  
Jia Yu ◽  
Yuxin Li ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Sh3 Domain ◽  
Dominant Negative ◽  
Β Cells ◽  
Β Cell ◽  
Inositol Hexakisphosphate ◽  
Endocytic Machinery ◽  
Molecular Machinery ◽  
The Individual ◽  
Syndrome Protein

Abstract Endocytosis is controlled by a well-orchestrated molecular machinery, where the individual players as well as their precise interactions are not fully understood. We now show that syndapin I/PACSIN 1 is expressed in pancreatic β cells and that its knockdown abrogates β cell endocytosis leading to disturbed plasma membrane protein homeostasis, as exemplified by an elevated density of L-type Ca2+ channels. Intriguingly, inositol hexakisphosphate (InsP6) activates casein kinase 2 (CK2) phosphorylating syndapin I/PACSIN 1, promoting interactions between syndapin I/PACSIN 1 and neural Wiskott-Aldrich syndrome protein (N-WASP) thereby driving β cell endocytosis. Dominant-negative interference with endogenous syndapin I/PACSIN 1 protein complexes, by overexpression of the syndapin I/PACSIN 1 SH3 domain, decreases InsP6-stimulated endocytosis. InsP6 thus promotes syndapin I/PACSIN 1 priming by CK2-dependent phosphorylation, which endows the syndapin I/PACSIN 1 SH3 domain with the capability to interact with the endocytic machinery and thereby initiate endocytosis, as exemplified in β cells.

scholarly journals Regulation of Insulin Secretion and β-Cell Mass by Activating Signal Cointegrator 2

2006 ◽  
Vol 26 (12) ◽  
pp. 4553-4563 ◽  
Author(s):  
Seon-Yong Yeom ◽  
Geun Hyang Kim ◽  
Chan Hee Kim ◽  
Heun Don Jung ◽  
So-Yeon Kim ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Endocrine Cells ◽  
Potential Role ◽  
Cell Mass ◽  
Dominant Negative ◽  
Transcriptional Coactivator ◽  
Β Cells ◽  
Β Cell ◽  
Islet Mass

ABSTRACT Activating signal cointegrator 2 (ASC-2) is a transcriptional coactivator of many nuclear receptors (NRs) and other transcription factors and contains two NR-interacting LXXLL motifs (NR boxes). In the pancreas, ASC-2 is expressed only in the endocrine cells of the islets of Langerhans, but not in the exocrine cells. Thus, we examined the potential role of ASC-2 in insulin secretion from pancreatic β-cells. Overexpressed ASC-2 increased glucose-elicited insulin secretion, whereas insulin secretion was decreased in islets from ASC-2+/− mice. DN1 and DN2 are two dominant-negative fragments of ASC-2 that contain NR boxes 1 and 2, respectively, and block the interactions of cognate NRs with the endogenous ASC-2. Primary rat islets ectopically expressing DN1 or DN2 exhibited decreased insulin secretion. Furthermore, relative to the wild type, ASC-2+/− mice showed reduced islet mass and number, which correlated with increased apoptosis and decreased proliferation of ASC-2+/− islets. These results suggest that ASC-2 regulates insulin secretion and β-cell survival and that the regulatory role of ASC-2 in insulin secretion appears to involve, at least in part, its interaction with NRs via its two NR boxes.

scholarly journals An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet β-cells

2010 ◽  
Vol 299 (1) ◽  
pp. E23-E32 ◽  
Author(s):  
Arthur T. Suckow ◽  
Branch Craige ◽  
Victor Faundez ◽  
William J. Cain ◽  
Steven D. Chessler
Keyword(s):  
Synaptic Vesicle ◽  
Pancreatic Islet ◽  
Membrane Trafficking ◽  
Protein Complexes ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Β Cells ◽  
Β Cell ◽  
Subunit Expression ◽  
Bona Fide

Pancreatic islet β-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since β-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in β-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates β-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 β-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits β3B and μ3B are expressed in β-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that β-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to β-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in β-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 δ-subunit expression. Our findings suggest that β-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.

scholarly journals MafA Is a Glucose-regulated and Pancreatic β-Cell-specific Transcriptional Activator for the Insulin Gene

2002 ◽  
Vol 277 (51) ◽  
pp. 49903-49910 ◽  
Author(s):  
Kohsuke Kataoka ◽  
Song-iee Han ◽  
Setsuko Shioda ◽  
Momoki Hirai ◽  
Makoto Nishizawa ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Regulatory Element ◽  
Transcriptional Activator ◽  
Dominant Negative ◽  
Insulin Gene ◽  
Pcr Analysis ◽  
Β Cells ◽  
Β Cell ◽  
Basic Leucine Zipper ◽  
Insulin Promoter

The insulin gene is specifically expressed in β-cells of the Langerhans islets of the pancreas, and its transcription is regulated by the circulating glucose level. Previous reports have shown that an unidentified β-cell-specific nuclear factor binds to a conservedcis-regulatory element called RIPE3b and is critical for its glucose-regulated expression. Based on the sequence similarity of the RIPE3b element and the consensus binding sequence of the Maf family of basic leucine zipper transcription factors, we here identified mammalianhomologueof avian MafA/L-Maf, an eye-specific member of the Maf family, as the RIPE3b-binding transcriptional activator. Reverse transcription-PCR analysis showed thatmafAmRNA is detected only in the eyes and in pancreatic β-cells and not in α-cells. MafA protein as well as its mRNA is up-regulated by glucose, consistent with the glucose-regulated binding of MafA to the RIPE3b element in β-cell nuclear extracts. In transient luciferase assays, we also showed that expression of MafA greatly enhanced insulin promoter activity and that a dominant-negative form of MafA inhibited it. Therefore, MafA is a β-cell-specific and glucose-regulated transcriptional activator for insulin gene expression and thus may be involved in the function and development of β-cells as well as in the pathogenesis of diabetes.

scholarly journals Glucocorticoids suppress β-cell development and induce hepatic metaplasia in embryonic pancreas

2003 ◽  
Vol 375 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Chia-Ning SHEN ◽  
Jonathan R. SECKL ◽  
Jonathan M. W. SLACK ◽  
David TOSH
Keyword(s):  
Transcription Factor ◽  
Fetal Programming ◽  
Later Life ◽  
Dominant Negative ◽  
Β Cells ◽  
Organ Cultures ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Embryonic Pancreas ◽  
Factor C

Elevated glucocorticoids are associated with low birth weight and fetal ‘programming’ of hypertension and glucose intolerance. In the present paper, we show that treatment of fetal rats with dexamethasone during the last week of gestation reduces the insulin content of their pancreatic β-cells. We reproduce this effect of dexamethasone in vitro using organ cultures of mouse embryonic pancreas, and show that it is associated with an elevation of expression of the transcription factor C/EBPβ (CCAAT/enhancer-binding protein β) and a reduction of the transcription factor Pdx-1 (pancreatic duodenal homeobox-1). Dexamethasone also induces the appearance of hepatocyte-like cells in organ cultures of pancreas, based on the expression of liver markers, albumin, α1-antitrypsin and transthyretin. Evidence that C/EBPβ is responsible for compromising the differentiation and later function of β-cells is obtained from its effects on the β-cell-like cell line RIN-5F. Transfection with a constitutive form of C/EBPβ suppresses insulin formation, whereas introduction of a dominant-negative inhibitor of C/EBPβ has no effect. We conclude that dexamethasone inhibits insulin expression in pancreatic β-cells via a mechanism involving down-regulation of Pdx-1 and induction of C/EBPβ. This mechanism may operate in combination with other changes during fetal programming, leading to type 2 diabetes in later life.

scholarly journals Identification of a WD40 Repeat-Containing Isoform of PHIP as a Novel Regulator of β-Cell Growth and Survival

2007 ◽  
Vol 27 (18) ◽  
pp. 6484-6496 ◽  
Author(s):  
Alexey Podcheko ◽  
Paul Northcott ◽  
George Bikopoulos ◽  
Andrew Lee ◽  
Swaroop R. Bommareddi ◽  
...  
Keyword(s):  
Cell Growth ◽  
Insulin Receptor ◽  
Cell Function ◽  
Strong Support ◽  
Dominant Negative ◽  
Pleckstrin Homology Domain ◽  
Β Cells ◽  
Β Cell ◽  
Wd40 Repeat ◽  
Cyclin D2

ABSTRACT The pleckstrin homology domain-interacting protein (PHIP) was originally identified as a 902-amino-acid (aa) protein that regulates insulin receptor-stimulated GLUT4 translocation in skeletal-muscle cells. Immunoblotting and immunohistological analyses of pancreatic β-cells reveal prominent expression of a 206-kDa PHIP isoform restricted to the nucleus. Herein, we report the cloning of this larger, 1,821-aa isoform of PHIP (PHIP1), which represents a novel WD40 repeat-containing protein. We demonstrate that PHIP1 overexpression stimulates insulin-like growth factor 1-dependent and -independent proliferation of β-cells, an event which correlates with transcriptional upregulation of the cyclin D2 promoter and the accumulation of cyclin D2 protein. RNA interference knockdown of PHIP1 in INS-1 cells abrogates insulin receptor substrate 2 (IRS2)-mediated DNA synthesis, providing for a specific role for PHIP1 in the enhancement of IRS2-dependent signaling responses leading to β-cell growth. Finally, we provide evidence that PHIP1 overexpression blocks free fatty acid-induced apoptosis in INS-1 cells, which is accompanied by marked activation of phosphoprotein kinase B (PKB)/AKT and the concomitant inhibition of caspase-9 and caspase-3 cleavage. Our finding that the restorative effect of PHIP1 on β-cell lipotoxicity can be attenuated by the overexpression of dominant-negative PKB suggests a key role for PKB in PHIP1-mediated cytoprotection. Taken together, these findings provide strong support for PHIP1 as a novel positive regulator of β-cell function. We suggest that PHIP1 may be involved in the induction of long-term gene expression programs to promote β-cell mitogenesis and survival.

scholarly journals Synaptosome-Associated Protein of 25 Kilodaltons Modulates Kv2.1 Voltage-Dependent K+ Channels in Neuroendocrine Islet β-Cells through an Interaction with the Channel N Terminus

2002 ◽  
Vol 16 (11) ◽  
pp. 2452-2461 ◽  
Author(s):  
Patrick E. MacDonald ◽  
Guotang Wang ◽  
Sharon Tsuk ◽  
Chikvashvili Dodo ◽  
Youhou Kang ◽  
...  
Keyword(s):  
Cell Voltage ◽  
Dominant Negative ◽  
K Channel ◽  
Β Cells ◽  
Β Cell ◽  
Syntaxin 1A ◽  
N Terminus ◽  
Voltage Dependent ◽  
K Currents

Abstract Insulin secretion is initiated by ionic events involving membrane depolarization and Ca2+ entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the β-cell voltage-dependent K+ channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K+ currents from rat β-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K+ currents in β-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other β-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.

scholarly journals Functional characterization of hyperpolarization-activated cyclic nucleotide-gated channels in rat pancreatic β cells

2009 ◽  
Vol 203 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Yi Zhang ◽  
Yunfeng Liu ◽  
Jihong Qu ◽  
Alexandre Hardy ◽  
Nina Zhang ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Membrane ◽  
Functional Characterization ◽  
Dominant Negative ◽  
Hcn Channels ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Over Expression

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate pacemaker activity in some cardiac cells and neurons. In the present study, we have identified the presence of HCN channels in pancreatic β-cells. We then examined the functional characterization of these channels in β-cells via modulating HCN channel activity genetically and pharmacologically. Voltage-clamp experiments showed that over-expression of HCN2 in rat β-cells significantly increased HCN current (Ih), whereas expression of dominant-negative HCN2 (HCN2-AYA) completely suppressed endogenous Ih. Compared to control β-cells, over-expression of Ih increased insulin secretion at 2.8 mmol/l glucose. However, suppression of Ih did not affect insulin secretion at both 2.8 and 11.1 mmol/l glucose. Current-clamp measurements revealed that HCN2 over-expression significantly reduced β-cell membrane input resistance (Rin), and resulted in a less-hyperpolarizing membrane response to the currents injected into the cell. Conversely, dominant negative HCN2-AYA expression led to a substantial increase of Rin, which was associated with a more hyperpolarizing membrane response to the currents injected. Remarkably, under low extracellular potassium conditions (2.5 mmol/l K+), suppression of Ih resulted in increased membrane hyperpolarization and decreased insulin secretion. We conclude that Ih in β-cells possess the potential to modulate β-cell membrane potential and insulin secretion under hypokalemic conditions.

scholarly journals Rhythm of the β-cell oscillator is not governed by a single regulator: multiple systems contribute to oscillatory behavior

2007 ◽  
Vol 292 (5) ◽  
pp. E1295-E1300 ◽  
Author(s):  
Emma Heart ◽  
Peter J. S. Smith
Keyword(s):  
Prolonged Exposure ◽  
Single Cells ◽  
Oscillatory Behavior ◽  
Glycolytic Flux ◽  
Β Cells ◽  
Β Cell ◽  
Multiple Systems ◽  
Oscillatory Pattern ◽  
Methyl Pyruvate ◽  
The Individual

Pulsatile insulin output, paralleled by oscillations in intracellular Ca2+, reflect oscillating metabolism within β-cells in response to secretory fuels. Here we question whether oscillatory periodicity is conserved or varied from stimulation to stimulation, whether glycolysis is essential for the manifestation of an oscillatory response, and if an environment of nutrient oversupply affects oscillatory regularity. We have determined that a β-cell oscillatory Ca2+ pattern is independent of the type of applied secretory fuel (glucose, methyl-pyruvate, or α-ketoisocaproate). In addition, single cells respond with the same pattern when repeatedly stimulated, regardless of the type of stimulatory fuel. Presence of substimulatory glucose is not necessary to obtain an oscillatory responses to methyl-pyruvate or α-ketoisocaproate. Glucose-6-phosphate, as a measure of glycolytic flux, is not detectable under these conditions. These data suggest that multiple systems, rather than a single enzyme component, can contribute to the β-cell oscillatory behavior. Prolonged exposure to high levels of palmitate impaired oscillatory regularity in the individual β-cells. This supports the hypothesis that a high-fat environment might contribute to loss of regular oscillatory pattern in diabetic subjects, acting, at least in part, at the level of the single β-cell.

Isolated mouse pancreatic β-cells show cell-specific temporal response pattern

2002 ◽  
Vol 282 (6) ◽  
pp. C1199-C1204 ◽  
Author(s):  
Gerd Larsson-Nyrén ◽  
Natalia Pakhtusova ◽  
Janove Sehlin
Keyword(s):  
Strong Correlation ◽  
Response Pattern ◽  
Lag Time ◽  
Temporal Response ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Lag Times ◽  
The Individual ◽  
Initial Peak

The length of the silent lag time before elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) differs between individual pancreatic β-cells. One important question is whether these differences reflect a random phenomenon or whether the length of lag time is inherent in the individual β-cell. We compared the lag times, initial dips, and initial peak heights for [Ca2+]i from two consecutive glucose stimulations (with either 10 or 20 mM glucose) in individual ob/ob mouse β-cells with the fura 2 technique in a microfluorimetric system. There was a strong correlation between the lengths of the lag times in each β-cell (10 mM glucose: r = 0.94, P < 0.001; 20 mM glucose: r = 0.96, P < 0.001) as well as between the initial dips in [Ca2+]i (10 mM glucose: r = 0.93, P < 0.001; 20 mM glucose: r = 0.79, P < 0.001) and between the initial peak heights (10 mM glucose: r = 0.51, P < 0.01; 20 mM glucose: r = 0.77, P < 0.001). These data provide evidence that the response pattern, including both the length of the lag time and the dynamics of the subsequent [Ca2+]i, is specific for the individual β-cell.

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