Syntaxin-6 SNARE Involvement in Secretory and Endocytic Pathways of Cultured Pancreatic β-Cells

Regina Kuliawat; Elena Kalinina; Jason Bock; Lloyd Fricker; Timothy E. McGraw; Se Ryoung Kim; Jiayu Zhong; Richard Scheller; Peter Arvan

doi:10.1091/mbc.e03-08-0554

Syntaxin-6 SNARE Involvement in Secretory and Endocytic Pathways of Cultured Pancreatic β-Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e03-08-0554 ◽

2004 ◽

Vol 15 (4) ◽

pp. 1690-1701 ◽

Cited By ~ 45

Author(s):

Regina Kuliawat ◽

Elena Kalinina ◽

Jason Bock ◽

Lloyd Fricker ◽

Timothy E. McGraw ◽

...

Keyword(s):

Secretory Granules ◽

Expression System ◽

Secretory Cells ◽

Β Cells ◽

Pancreatic Β Cells ◽

Rate Limiting Step ◽

Protein Receptor ◽

Golgi Network ◽

Rate Limiting

In pancreatic β-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 β-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of β-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.

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Pancreatic β-Cell Protein Granuphilin Binds Rab3 and Munc-18 and Controls Exocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.02-02-0025 ◽

2002 ◽

Vol 13 (6) ◽

pp. 1906-1915 ◽

Cited By ~ 104

Author(s):

Thierry Coppola ◽

Christian Frantz ◽

Véronique Perret-Menoud ◽

Sonia Gattesco ◽

Harald Hirling ◽

...

Keyword(s):

Binding Protein ◽

Secretory Granules ◽

Secretory Vesicle ◽

Cell Protein ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Protein Receptor

Granuphilin/Slp-4 is a member of the synaptotagmin-like protein family expressed in pancreatic β-cells and in the pituitary gland. We show by confocal microscopy that both granuphilin-a and -b colocalize with insulin-containing secretory granules positioned at the periphery of pancreatic β-cells. Overexpression of granuphilins in insulin-secreting cell lines caused a profound inhibition of stimulus-induced exocytosis. Granuphilins were found to bind to two components of the secretory machinery of pancreatic β-cells, the small GTP-binding protein Rab3 and the solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)–binding protein Munc-18. The interaction with Rab3 occurred only with the GTP-bound form of the protein and was prevented by a point mutation in the effector domain of the GTPase. Structure-function studies using granuphilin-b mutants revealed that complete loss of Rab3 binding is associated with a reduction in the capacity to inhibit exocytosis. However, the granuphilin/Rab3 complex alone is not sufficient to mediate the decrease of exocytosis, suggesting the existence of additional binding partners. Taken together, our observations indicate that granuphilins play an important role in pancreatic β-cell exocytosis. In view of the postulated role of Munc-18 in secretory vesicle docking, our data suggest that granuphilins may also be involved in this process.

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The role of calcium in the regulation of renin secretion

AJP Renal Physiology ◽

10.1152/ajprenal.00143.2009 ◽

2010 ◽

Vol 298 (1) ◽

pp. F1-F11 ◽

Cited By ~ 53

Author(s):

William H. Beierwaltes

Keyword(s):

Intracellular Calcium ◽

Second Messenger ◽

Renin Secretion ◽

Secretory Cells ◽

The Past ◽

Calcium Sensing ◽

Rate Limiting Step ◽

Rate Limiting ◽

Synthesis And Degradation

Renin is the enzyme which is the rate-limiting step in the formation of the hormone angiotensin II. Therefore, the regulation of renin secretion is critical in understanding the control of the renin-angiotensin-aldosterone system and its many biological and pathological actions. Renin is synthesized, stored in, and released from the juxtaglomerular (JG) cells of the kidney. While renin secretion is positively regulated by the “second messenger” cAMP, unlike most secretory cells, renin secretion from the JG cell is inversely related to the extracellular and intracellular calcium concentrations. This novel relationship is referred to as the “calcium paradox.” This review will address observations made over the past 30 years regarding calcium and the regulation of renin secretion, and focus on recent observations which address this scientific conundrum. These include 1) receptor-mediated pathways for changing intracellular calcium; 2) the discovery of a calcium-inhibitable isoform of adenylyl cyclase associated with renin in the JG cells; 3) calcium-sensing receptors in the JG cells; 4) calcium-calmodulin-mediated signals; 5) the role of phosphodiesterases; and 6) connexins, gap junctions, calcium waves, and the cortical extracellular calcium environment. While cAMP is the dominant second messenger for renin secretion, calcium appears to modulate the integrated activities of the enzymes, which balance cAMP synthesis and degradation. Thus this review concludes that calcium modifies the amplitude of cAMP-mediated renin-signaling pathways. While calcium does not directly control renin secretion, increased calcium inhibits and decreased calcium amplifies cAMP-stimulated renin secretion.

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Requirement for N-ethylmaleimide-sensitive factor for exocytosis of insulin-containing secretory granules in pancreatic β-cells

Biochemical Society Transactions ◽

10.1042/bst0310842 ◽

2003 ◽

Vol 31 (4) ◽

pp. 842-847 ◽

Cited By ~ 3

Author(s):

J. Vikman ◽

X. Ma ◽

M. Tagaya ◽

L. Eliasson

Keyword(s):

Secretory Granules ◽

Β Cells ◽

Pancreatic Β Cells ◽

Exact Role ◽

Capacitance Measurements ◽

Reserve Pool ◽

Sensitive Factor ◽

Intracellular Compartments ◽

Rat Insulinoma

N-Ethylmaleimide-sensitive factor (NSF) has an important role in fusion processes within intracellular compartments and at the plasma membrane, but the exact role of this protein in the exocytotic machinery has not yet been determined. NSF was found to be present in the cytosol of rat pancreatic β-cells and rat insulinoma INS-1 cells. Capacitance measurements revealed that exocytosis of primed granules was not affected by the presence of a monoclonal antibody against NSF, mAb 2E5, suggesting that NSF is not involved in the fusion process. The antibody markedly decreased rapid refilling of new granules from a reserve pool during a first stimulation. However, slow refilling of primed granules occurred within a 2 min period between the first and second stimulations. We conclude that NSF is required in the exocytotic process in order to obtain a complete exocytotic response. Possible mechanisms by which NSF takes part in this process in insulin-secreting rat β-cells are discussed.

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Inside the Insulin Secretory Granule

Metabolites ◽

10.3390/metabo11080515 ◽

2021 ◽

Vol 11 (8) ◽

pp. 515

Author(s):

Mark Germanos ◽

Andy Gao ◽

Matthew Taper ◽

Belinda Yau ◽

Melkam A. Kebede

Keyword(s):

Secretory Granules ◽

Β Cells ◽

Β Cell ◽

Proinsulin Processing ◽

Membrane Bound ◽

Golgi Network ◽

Insulin Secretory Granule ◽

Insulin Storage ◽

Cellular Stimulation

The pancreatic β-cell is purpose-built for the production and secretion of insulin, the only hormone that can remove glucose from the bloodstream. Insulin is kept inside miniature membrane-bound storage compartments known as secretory granules (SGs), and these specialized organelles can readily fuse with the plasma membrane upon cellular stimulation to release insulin. Insulin is synthesized in the endoplasmic reticulum (ER) as a biologically inactive precursor, proinsulin, along with several other proteins that will also become members of the insulin SG. Their coordinated synthesis enables synchronized transit through the ER and Golgi apparatus for congregation at the trans-Golgi network, the initiating site of SG biogenesis. Here, proinsulin and its constituents enter the SG where conditions are optimized for proinsulin processing into insulin and subsequent insulin storage. A healthy β-cell is continually generating SGs to supply insulin in vast excess to what is secreted. Conversely, in type 2 diabetes (T2D), the inability of failing β-cells to secrete may be due to the limited biosynthesis of new insulin. Factors that drive the formation and maturation of SGs and thus the production of insulin are therefore critical for systemic glucose control. Here, we detail the formative hours of the insulin SG from the luminal perspective. We do this by mapping the journey of individual members of the SG as they contribute to its genesis.

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Pancreatic β-cells express hepcidin, an iron-uptake regulatory peptide

Journal of Endocrinology ◽

10.1677/joe-07-0528 ◽

2008 ◽

Vol 197 (2) ◽

pp. 241-249 ◽

Cited By ~ 50

Author(s):

Hasan Kulaksiz ◽

Evelyn Fein ◽

Peter Redecker ◽

Wolfgang Stremmel ◽

Guido Adler ◽

...

Keyword(s):

Iron Uptake ◽

Secretory Granules ◽

Rinm5f Cells ◽

Β Cells ◽

Additional Source ◽

Β Cell ◽

Pancreatic Β Cells ◽

Vital Functions ◽

Immunohistochemical Studies ◽

Insight Into

Body iron is involved in various vital functions. Its uptake in the intestine is regulated by hepcidin, a bioactive peptide originally identified in plasma and urine and subsequently in the liver. In the present study, we provide evidence at the transcriptional and translational levels that hepcidin is also expressed in the pancreas of rat and man. Immunohistochemical studies localized the peptide exclusively to β-cells of the islets of Langerhans. Immunoelectron microscopical analyses revealed that hepcidin is confined to the insulin-storing β-cell secretory granules. As demonstrated in insulinoma-derived RINm5F cells, the expression of hepcidin in β-cells is regulated by iron. Based on the present findings we conclude that pancreatic islets are an additional source of the peptide hepcidin. The localization of this peptide to β-cells suggests that pancreatic β-cells may be involved in iron metabolism in addition to their genuine function in blood glucose regulation. In view of the various linked iron/glucose disorders in the pancreas, the present findings may provide an insight into the phenomenology of intriguing mutual relationships between iron and glucose metabolisms.

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Conditional expression of the FTO gene product in rat INS-1 cells reveals its rapid turnover and a role in the profile of glucose-induced insulin secretion

Clinical Science ◽

10.1042/cs20100416 ◽

2011 ◽

Vol 120 (9) ◽

pp. 403-413 ◽

Cited By ~ 15

Author(s):

Mark A. Russell ◽

Noel G. Morgan

Keyword(s):

Insulin Secretion ◽

Expression System ◽

Proteasome Inhibitors ◽

Inducible Promoter ◽

Insulin Biosynthesis ◽

Second Phase ◽

Β Cells ◽

Pancreatic Β Cells ◽

Peripheral Tissues ◽

Conditional Expression

Common polymorphisms within the FTO (fat mass and obesity-associated) gene correlate with increased BMI (body mass index) and a rising risk of Type 2 diabetes. FTO is highly expressed in the brain but has also been detected in peripheral tissues, including the endocrine pancreas, although its function there is unclear. The aim of the present study was to investigate the role of FTO protein in pancreatic β-cells using a conditional expression system developed in INS-1 cells. INS-1 cells were stably transfected with FTO–HA (haemagluttinin) incorporated under the control of a tetracycline-inducible promoter. Induction of FTO protein resulted in localization of the tagged protein to the nucleus. The level of FTO–HA protein achieved in transfected cells was tightly regulated, and experiments with selective inhibitors revealed that FTO–HA is rapidly degraded via the ubiquitin/proteasome pathway. The nuclear localization was not altered by proteasome inhibitors, although following treatment with PYR-41, an inhibitor of ubiquitination, some of the protein adopted a perinuclear localization. Unexpectedly, modestly increased expression of FTO–HA selectively enhanced the first phase of insulin secretion when INS-1 monolayers or pseudoislets were stimulated with 20 mM glucose, whereas the second phase remained unchanged. The mechanism responsible for the potentiation of glucose-induced insulin secretion is unclear; however, further experiments revealed that it did not involve an increase in insulin biosynthesis or any changes in STAT3 (signal transducer and activator of transcription 3) expression. Taken together, these results suggest that the FTO protein may play a hitherto unrecognized role in the control of first-phase insulin secretion in pancreatic β-cells.

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