Synaptotagmin III/VII Isoforms Mediate Ca2+-induced Insulin Secretion in Pancreatic Islet β-Cells
2000; Elsevier BV; Volume: 275; Issue: 46 Linguagem: Inglês
10.1074/jbc.m004284200
ISSN1083-351X
AutoresZhiyong Gao, John Reavey‐Cantwell, Robert Young, Patricia Jegier, Bryan A. Wolf,
Tópico(s)Adenosine and Purinergic Signaling
ResumoSynaptotagmins (Syt) play important roles in Ca2+-induced neuroexocytosis. Insulin secretion of the pancreatic β-cell is dependent on an increase in intracellular Ca2+; however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting βTC3 cell. Syt III and VII proteins were identified in rat islets and βTC3 and RINm5F β-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F β-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca2+ sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca2+-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F β-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca2+-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca2+sensor or one of the Ca2+ sensors for insulin exocytosis of the β-cell. Synaptotagmins (Syt) play important roles in Ca2+-induced neuroexocytosis. Insulin secretion of the pancreatic β-cell is dependent on an increase in intracellular Ca2+; however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting βTC3 cell. Syt III and VII proteins were identified in rat islets and βTC3 and RINm5F β-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F β-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca2+ sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca2+-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F β-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca2+-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca2+sensor or one of the Ca2+ sensors for insulin exocytosis of the β-cell. synaptotagmin βTC3 insulin-secreting β-cell C-terminal peptide of synaptotagmin III C-terminal peptide of synaptotagmin VII RINm5F β-cell empty vector-transfected RINm5F β-cell bovine serum albumin reverse transcriptase-polymerase chain reaction 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid guanosine 5′-3-O-(thio)triphosphate phosphate-buffered saline Insulin exocytosis from the β-cell of the islets of Langerhans is stimulated by various physiological secretagogues that include glucose, amino acids, and receptor-mediated agonists such as acetylcholine, cholecystokinin, and glucagon like-peptide 1 (1Easom R.A. Diabetes. 1999; 48: 675-684Crossref PubMed Scopus (104) Google Scholar, 2Hedeskov C.J. Physiol. Rev. 1980; 60: 442-509Crossref PubMed Scopus (326) Google Scholar, 3Henquin J.C. Jonas J.C. Gilon P. Diabetes Metab. 1998; 24: 30-36PubMed Google Scholar, 4Matschinsky F.M. Diabetes. 1996; 45: 223-241Crossref PubMed Scopus (0) Google Scholar, 5Polonsky K.S. Diabetes. 1995; 44: 705-717Crossref PubMed Google Scholar, 6Prentki M. Tornheim K. Corkey B.E. Diabetologia. 1997; 40 (suppl.): S32-S41Crossref Scopus (151) Google Scholar, 7Wollheim C.B. Regazzi R. FEBS Lett. 1990; 268: 376-380Crossref PubMed Scopus (61) Google Scholar). A common mechanism of action for these secretagogues is to cause an increase in cytosolic Ca2+. Elevation of intracellular Ca2+ is due to an influx of extracellular Ca2+through voltage-dependent L-type Ca2+ channel and/or mobilization of intracellular Ca2+ from the endoplasmic reticulum (8Wollheim C.B. Kikuchi M. Renold A.E. Sharp G.W. J. Clin. Invest. 1978; 62: 451-458Crossref PubMed Scopus (81) Google Scholar, 9Prentki M. Wollheim C.B. Experientia (Basel). 1984; 40: 1052-1060Crossref PubMed Scopus (64) Google Scholar, 10Turk J. Wolf B.A. McDaniel M.L. Prog. Lipid Res. 1987; 26: 125-181Crossref PubMed Scopus (115) Google Scholar, 11Wolf B.A. Colca J.R. Turk J. Florholmen J. McDaniel M.L. Am. J. Physiol. 1988; 254: E121-E136PubMed Google Scholar, 12Hellman B. Gylfe E. Grapengiesser E. Lund P.E. Berts A. Biochim. Biophys. Acta. 1992; 1113: 295-305Crossref PubMed Scopus (142) Google Scholar, 13Rasmussen H. Isales C.M. Calle R. Throckmorton D. Anderson M. Gasalla-Herraiz J. McCarthy R. Endocr. Rev. 1995; 16: 649-681PubMed Google Scholar, 14Komatsu M. Schermerhorn T. Noda M. Straub S.G. Aizawa T. Sharp G.W. Diabetes. 1997; 46: 1928-1938Crossref PubMed Scopus (93) Google Scholar, 15Aspinwall C.A. Lakey J.R. Kennedy R.T. J. Biol. Chem. 1999; 274: 6360-6365Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 16Lang J. Eur. J. Biochem. 1999; 259: 3-17Crossref PubMed Scopus (279) Google Scholar, 17Xu G.G. Gao Z. Borge Jr., P.D. Wolf B.A. J. Biol. Chem. 1999; 274: 18067-18074Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). However, the mechanisms by which Ca2+ induces insulin granule fusion with the plasma membrane of β-cell remain unclear (1Easom R.A. Diabetes. 1999; 48: 675-684Crossref PubMed Scopus (104) Google Scholar, 16Lang J. Eur. J. Biochem. 1999; 259: 3-17Crossref PubMed Scopus (279) Google Scholar, 18Daniel S. Noda M. Straub S.G. Sharp G.W. Diabetes. 1999; 48: 1686-1690Crossref PubMed Scopus (173) Google Scholar, 20Wiser O. Trus M. Hernandez A. Renstrom E. Barg S. Rorsman P. Atlas D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 248-253Crossref PubMed Scopus (246) Google Scholar, 21Pouli A.E. Emmanouilidou E. Zhao C. Wasmeier C. Hutton J.C. Rutter G.A. Biochem. J. 1998; 333: 193-199Crossref PubMed Scopus (124) Google Scholar).Synaptotagmin (Syt)1 is a family of membrane proteins initially found to be expressed in brain. At the present, 11 members of Syt have been identified (22Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar, 23Sudhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). The Syt molecule has a single transmembrane domain and two Ca2+regulatory C2 domains. The C2 domains mediate Ca2+-dependent and Ca2+-independent interactions with target molecules that may regulate membrane fusion and membrane budding reactions (24Sugita S. Sudhof T.C. Biochemistry. 2000; 39: 2940-2949Crossref PubMed Scopus (50) Google Scholar, 25Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Literature concerning the expression and functions of Syt in pancreatic β-cell is very limited and contradictory. In an earlier study (26Jacobsson G. Bean A.J. Scheller R.H. Juntti-Berggren L. Deeney J.T. Berggren P.O. Meister B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12487-12491Crossref PubMed Scopus (194) Google Scholar), Syt was found in the non-β-cell of the islet mantle, but not in the β-cell, using a non-isoform-specific antibody, and the mRNAs of Syt A and B were absent in mouse pancreatic β-cell and RINm5F cells as demonstrated byin situ hybridization. Recently, the mRNA and protein of Syt isoforms I and II (27Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (101) Google Scholar) were found in insulin-secreting β-cell lines RINm5F, INS-1, and HIT-T15. It was reported that Syt I, II (27Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (101) Google Scholar), and Syt III proteins (28Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar) are localized mainly in insulin-containing secretory granules. However, only the mRNA but not the protein was detected in primary islet β-cells (27Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (101) Google Scholar). In other studies, Syt III mRNA was present in MIN6, RINm5F, HIT-T15, and βTC6-f7 (29Wheeler M.B. Sheu L. Ghai M. Bouquillon A. Grondin G. Weller U. Beaudoin A.R. Bennett M.K. Trimble W.S. Gaisano H.Y. Endocrinology. 1996; 137: 1340-1348Crossref PubMed Scopus (182) Google Scholar) cells and pancreatic islets (28Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar), and the protein expression of Syt III in MIN6 cell and pancreatic islets was confirmed by one group (28Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar) but not by another (29Wheeler M.B. Sheu L. Ghai M. Bouquillon A. Grondin G. Weller U. Beaudoin A.R. Bennett M.K. Trimble W.S. Gaisano H.Y. Endocrinology. 1996; 137: 1340-1348Crossref PubMed Scopus (182) Google Scholar). The aims of the current study were to examine the expression of various Syt isoforms in pancreatic islets as well as insulin-secreting β-cell lines, the subcellular localization of Syt, and the functional role of Syt in insulin exocytosis using a β-cell line overexpressing Syt.DISCUSSIONIncreased cytosolic Ca2+ is required for secretagogue-induced insulin secretion from pancreatic β-cells. The various isoforms of synaptotagmins are known to play major roles in regulated secretion of neurotransmitters in presynaptic terminals (22Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar,23Sudhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Synaptotagmin has been shown to bind Ca2+ in a phospholipid-dependent manner and undergoes a Ca2+-dependent conformational change that enables it to bind syntaxin on the plasma membrane for granule fusion. The expression of Syt I and II mRNA and protein in pancreatic β-cells has recently been shown in insulin-secreting β-cell lines RINm5F, INS-1, and HIT-T15 (27Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (101) Google Scholar). An involvement of Syt I or II in insulin secretion of tumor cell lines is supported by the inhibition of Ca2+-induced insulin secretion in permeabilized cells using antibodies directed against the Ca2+-dependent phospholipid-binding site of the first C2 domain of Syt I or II. However, overexpression of Syt II in HIT-T15 β-cell failed to affect insulin secretion (27Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (101) Google Scholar). Moreover, this hypothesis may not be applicable to normal pancreatic β-cells, because of the lack of Syt I and II expression in primary islet β-cell as shown previously and in this study (27Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (101) Google Scholar). These observations suggest that Syt I and II do not have a physiological role in insulin secretion and imply the presence of alternative signaling Syt proteins in the pancreatic β-cell. Another argument against the role of Syt I in insulin secretion is that it requires levels of free Ca2+ that are at least 1 to 2 orders of magnitude higher than those observed during insulin exocytosis in the β-cells (22Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar, 45Littleton J.T. Bellen H.J. Trends Neurosci. 1995; 18: 177-183Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 46Sutton R.B. Davletov B.A. Berghuis A.M. Sudhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (602) Google Scholar).Our study has discovered new Syt isoforms (IV, V, VII, and VIII) as well as confirmed previously reported Syt isoform (I, II and III) expression in βTC3 (I, II, III, IV, V, VII, and VIII), RINm5F cells (III and VII), and rat primary islet β-cells (III, IV, V, and VII). The expression of Syt VII protein in βTC3, RIMm5F, and primary rat islet is a novel finding. The absence of RT-PCR product for synaptotagmin isoforms I, II, VI, and VIII in primary rat islets suggests they are either not expressed or expressed at very low levels. The difference of isoform expression between tumor cell line and primary rat islet cell cannot be easily explained. However, this may indicate that isoforms III and VII are more physiologically important than isoforms I and II in insulin secretion. Further investigation is required to address the roles of Syt IV and V in pancreatic β-cell.An important candidate for insulin exocytosis is the Syt III isoform (47Brown H. Meister B. Deeney J. Corkey B.E. Yang S.N. Larsson O. Rhodes C.J. Seino S. Berggren P.O. Fried G. Diabetes. 2000; 49: 383-391Crossref PubMed Scopus (44) Google Scholar, 48Mizuta M. Kurose T. Miki T. Shoji-Kasai Y. Takahashi M. Seino S. Matsukura S. Diabetes. 1997; 46: 2002-2006Crossref PubMed Google Scholar). Its Ca2+ sensitivity is submicromolar, which is the physiological range present in stimulated β-cells. Previous studies have mainly focused on Syt III identification in β-cells. Syt III has been found in the insulin-secreting cell line MIN6 as well as in primary islet cells (48Mizuta M. Kurose T. Miki T. Shoji-Kasai Y. Takahashi M. Seino S. Matsukura S. Diabetes. 1997; 46: 2002-2006Crossref PubMed Google Scholar). Syt III and VII have been localized to insulin granules in various β-cell lines and islet cells, which implicates that they may play important roles in granule fusion and exocytosis (47Brown H. Meister B. Deeney J. Corkey B.E. Yang S.N. Larsson O. Rhodes C.J. Seino S. Berggren P.O. Fried G. Diabetes. 2000; 49: 383-391Crossref PubMed Scopus (44) Google Scholar). Our study shows that by confocal microscopy, synaptotagmin III and VII co-localize with insulin secretory granules. Thus, all studies so far have localized Syt III in the β-cell and in particular to the insulin-containing secretory granule.Because of the localization of Syt III and Syt VII to the insulin secretory granule, it is conceivable that they may have a major role in insulin exocytosis, by analogy with their known role in neurotransmitter release. Treatment of permeabilized MIN6 cells with anti-Syt III antibody inhibited Ca2+-triggered insulin secretion (48Mizuta M. Kurose T. Miki T. Shoji-Kasai Y. Takahashi M. Seino S. Matsukura S. Diabetes. 1997; 46: 2002-2006Crossref PubMed Google Scholar). Another study reported that synaptotagmin III antibodies inhibited Ca2+-induced changes in β-cell membrane capacitance (47Brown H. Meister B. Deeney J. Corkey B.E. Yang S.N. Larsson O. Rhodes C.J. Seino S. Berggren P.O. Fried G. Diabetes. 2000; 49: 383-391Crossref PubMed Scopus (44) Google Scholar). Although indirect, these experiments implicate a role of Syt III in insulin secretion. We have directly addressed this issue by engineering insulin-secreting β-cells that overexpress Syt III or Syt VII and compared them to an empty vector Neo control β-cell line.Our results clearly show that β-cells overexpressing Syt III secrete more insulin in response to stimuli that increase intracellular Ca2+. This effect was consistently observed in intact β-cells as well as permeabilized β-cells. In normal β-cells, depolarization with K+ causes an influx of extracellular Ca2+ and subsequent insulin secretion. However, in the β-cells overexpressing Syt III, K+-induced insulin secretion was further amplified 6-fold. In the case of glyceraldehyde- and carbachol-induced insulin secretion, the amplification obtained was only 2.5-fold. This is probably due to the fact that cytosolic Ca2+ elevation is the sole mechanism for K+-induced insulin secretion, whereas glyceraldehyde and carbachol stimulate insulin secretion through increased intracellular Ca2+ as well as other mechanisms, such as activation of protein kinase C (8Wollheim C.B. Kikuchi M. Renold A.E. Sharp G.W. J. Clin. Invest. 1978; 62: 451-458Crossref PubMed Scopus (81) Google Scholar, 9Prentki M. Wollheim C.B. Experientia (Basel). 1984; 40: 1052-1060Crossref PubMed Scopus (64) Google Scholar, 10Turk J. Wolf B.A. McDaniel M.L. Prog. Lipid Res. 1987; 26: 125-181Crossref PubMed Scopus (115) Google Scholar, 11Wolf B.A. Colca J.R. Turk J. Florholmen J. McDaniel M.L. Am. J. Physiol. 1988; 254: E121-E136PubMed Google Scholar, 12Hellman B. Gylfe E. Grapengiesser E. Lund P.E. Berts A. Biochim. Biophys. Acta. 1992; 1113: 295-305Crossref PubMed Scopus (142) Google Scholar, 13Rasmussen H. Isales C.M. Calle R. Throckmorton D. Anderson M. Gasalla-Herraiz J. McCarthy R. Endocr. Rev. 1995; 16: 649-681PubMed Google Scholar, 14Komatsu M. Schermerhorn T. Noda M. Straub S.G. Aizawa T. Sharp G.W. Diabetes. 1997; 46: 1928-1938Crossref PubMed Scopus (93) Google Scholar, 15Aspinwall C.A. Lakey J.R. Kennedy R.T. J. Biol. Chem. 1999; 274: 6360-6365Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 16Lang J. Eur. J. Biochem. 1999; 259: 3-17Crossref PubMed Scopus (279) Google Scholar, 17Xu G.G. Gao Z. Borge Jr., P.D. Wolf B.A. J. Biol. Chem. 1999; 274: 18067-18074Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Similarly, the lack of amplification of glyceraldehyde-induced secretion in Syt III β-cells is probably due to the even lower contribution of Ca2+-dependent signaling in this pathway. Increased Ca2+-induced insulin secretion was also directly observed in permeabilized Syt III β-cells. Mastoparan, which directly triggers insulin granule fusion and exocytosis (33Konrad R.J. Young R.A. Record R.D. Smith R.M. Butkerait P. Manning D. Jarett L. Wolf B.A. J. Biol. Chem. 1995; 270: 12869-12876Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), also increased insulin secretion. The findings in our study suggest that Syt III and Syt VII may be the Ca2+ sensor or one of the Ca2+ sensors for insulin exocytosis of the β-cell and may be a potentially interesting target for cellular and pharmacological therapies aimed at increasing insulin secretion. The possibility that Syt IV or V isoforms play a role in Ca2+ sensing for insulin exocytosis cannot be excluded.It is interesting that both Syt III and Syt VII β-cells had increased insulin secretion induced by direct elevation of intracellular Ca2+ concentration in permeabilized cells. However, they secreted insulin differently in response to carbachol and high K+. The amplified response to 30 mmK+ was larger in Syt III cells and not significant in Syt VII cells. On the other hand, insulin secretion triggered by carbachol was larger in Syt VII cells but was not significant in Syt III cells. The explanation of these differences is currently unclear. One possibility is that the two Syt isoforms sense changes of cytosolic Ca2+ in different subcellular locations. For example, Syt III may sense an increase in intracellular Ca2+ due to influx of extracellular Ca2+, whereas Syt VII may be relevant when intracellular Ca2+ elevation is induced by intracellular mobilization through inositol 1,4,5-trisphosphate receptors. It has been demonstrated that synaptotagmin directly interacts with N-type (49Leveque C. Hoshino T. David P. Shoji-Kasai Y. Leys K. Omori A. Lang B. El Far O. Sato K. Martin-Moutot N. Newsom-Davis J. Takahashi M. Seagar M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3625-3629Crossref PubMed Scopus (190) Google Scholar, 50O'Connor V.M. Shamotienko O. Grishin E. Betz H. FEBS Lett. 1993; 326: 255-260Crossref PubMed Scopus (136) Google Scholar, 51David P. Martin-Moutot N. Leveque C. El Far O. Takahashi M. Seagar M.J. Neuromuscular Disorders. 1993; 3: 451-454Abstract Full Text PDF PubMed Scopus (7) Google Scholar, 52Lévêque C. el Far O. Martin-Moutot N. Sato K. Kato R. Takahashi M. Seagar M.J. J. Biol. 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Inositol 1,4,5-trisphosphate receptors are present in pancreatic β-cells; however, it is currently unclear whether they associate with synaptotagmin.In conclusion, we have shown that isoforms III, IV, V, and VII of synaptotagmin instead of isoforms I and II are expressed in islet β-cells and that Syt III and Syt VII may have an important physiological role in insulin exocytosis of the pancreatic β-cell. The physiological roles of the other isoforms remain to be investigated. Insulin exocytosis from the β-cell of the islets of Langerhans is stimulated by various physiological secretagogues that include glucose, amino acids, and receptor-mediated agonists such as acetylcholine, cholecystokinin, and glucagon like-peptide 1 (1Easom R.A. Diabetes. 1999; 48: 675-684Crossref PubMed Scopus (104) Google Scholar, 2Hedeskov C.J. Physiol. Rev. 1980; 60: 442-509Crossref PubMed Scopus (326) Google Scholar, 3Henquin J.C. Jonas J.C. Gilon P. Diabetes Metab. 1998; 24: 30-36PubMed Google Scholar, 4Matschinsky F.M. Diabetes. 1996; 45: 223-241Crossref PubMed Scopus (0) Google Scholar, 5Polonsky K.S. Diabetes. 1995; 44: 705-717Crossref PubMed Google Scholar, 6Prentki M. Tornheim K. Corkey B.E. Diabetologia. 1997; 40 (suppl.): S32-S41Crossref Scopus (151) Google Scholar, 7Wollheim C.B. Regazzi R. FEBS Lett. 1990; 268: 376-380Crossref PubMed Scopus (61) Google Scholar). A common mechanism of action for these secretagogues is to cause an increase in cytosolic Ca2+. Elevation of intracellular Ca2+ is due to an influx of extracellular Ca2+through voltage-dependent L-type Ca2+ channel and/or mobilization of intracellular Ca2+ from the endoplasmic reticulum (8Wollheim C.B. Kikuchi M. Renold A.E. Sharp G.W. J. Clin. Invest. 1978; 62: 451-458Crossref PubMed Scopus (81) Google Scholar, 9Prentki M. Wollheim C.B. Experientia (Basel). 1984; 40: 1052-1060Crossref PubMed Scopus (64) Google Scholar, 10Turk J. Wolf B.A. McDaniel M.L. Prog. 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Chem. 1999; 274: 18067-18074Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). However, the mechanisms by which Ca2+ induces insulin granule fusion with the plasma membrane of β-cell remain unclear (1Easom R.A. Diabetes. 1999; 48: 675-684Crossref PubMed Scopus (104) Google Scholar, 16Lang J. Eur. J. Biochem. 1999; 259: 3-17Crossref PubMed Scopus (279) Google Scholar, 18Daniel S. Noda M. Straub S.G. Sharp G.W. Diabetes. 1999; 48: 1686-1690Crossref PubMed Scopus (173) Google Scholar, 20Wiser O. Trus M. Hernandez A. Renstrom E. Barg S. Rorsman P. Atlas D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 248-253Crossref PubMed Scopus (246) Google Scholar, 21Pouli A.E. Emmanouilidou E. Zhao C. Wasmeier C. Hutton J.C. Rutter G.A. Biochem. J. 1998; 333: 193-199Crossref PubMed Scopus (124) Google Scholar). Synaptotagmin (Syt)1 is a family of membrane proteins initially found to be expressed in brain. At the present, 11 members of Syt have been identified (22Sudhof T.C. 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However, only the mRNA but not the protein was detected in primary islet β-cells (27Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (101) Google Scholar). In other studies, Syt III mRNA was present in MIN6, RINm5F, HIT-T15, and βTC6-f7 (29Wheeler M.B. Sheu L. Ghai M. Bouquillon A. Grondin G. Weller U. Beaudoin A.R. Bennett M.K. Trimble W.S. Gaisano H.Y. Endocrinology. 1996; 137: 1340-1348Crossref PubMed Scopus (182) Google Scholar) cells and pancreatic islets (28Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar), and the protein expression of Syt III in MIN6 cell and pancreatic islets was confirmed by one group (28Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar) but not by another (29Wheeler M.B. Sheu L. Ghai M. Bouquillon A. Grondin G. Weller U. Beaudoin A.R. Bennett M.K. Trimble W.S. Gaisano H.Y. Endocrinology. 1996; 137: 1340-1348Crossref PubMed Scopus (182) Google Scholar). The aims of the current study were to examine the expression of various Syt isoforms in pancreatic islets as well as insulin-secreting β-cell lines, the subcellular localization of Syt, and the functional role of Syt in insulin exocytosis using a β-cell line overexpressing Syt. DISCUSSIONIncreased cytosolic Ca2+ is required for secretagogue-induced insulin secretion from pancreatic β-cells. The various isoforms of synaptotagmins are known to play major roles in regulated secretion of neurotransmitters in presynaptic terminals (22Sudhof T.C. Nature. 1995; 375: 645-653Cro
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