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Roles of Insulin Receptor Substrate-1, Phosphatidylinositol 3-Kinase, and Release of Intracellular Ca2+ Stores in Insulin-stimulated Insulin Secretion in β-Cells*

Open AccessPublished:July 21, 2000DOI:https://doi.org/10.1074/jbc.M909647199
      The signaling pathway by which insulin stimulates insulin secretion and increases in intracellular free Ca2+ concentration ([Ca2+]i) in isolated mouse pancreatic β-cells and clonal β-cells was investigated. Application of insulin to single β-cells resulted in increases in [Ca2+]i that were of lower magnitude, slower onset, and longer lifetime than that observed with stimulation with tolbutamide. Furthermore, the increases in [Ca2+]i originated from interior regions of the cell rather than from the plasma membrane as with depolarizing stimuli. The insulin-induced [Ca2+]i changes and insulin secretion at single β-cells were abolished by treatment with 100 nm wortmannin or 1 μm thapsigargin; however, they were unaffected by 10 μm U73122, 20 μmnifedipine, or removal of Ca2+ from the medium. Insulin-stimulated insulin secretion was also abolished by treatment with 2 μm bisindolylmaleimide I, but [Ca2+]i changes were unaffected. In an insulin receptor substrate-1 gene disrupted β-cell tumor line, insulin did not evoke either [Ca2+]i changes or insulin secretion. The data suggest that autocrine-activated increases in [Ca2+]i are due to release of intracellular Ca2+ stores, especially the endoplasmic reticulum, mediated by insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Autocrine activation of insulin secretion is mediated by the increase in [Ca2+]i and activation of protein kinase C.
      IRS
      insulin receptor substrate
      PI3-K
      phosphatidylinositol 3-kinase
      PIP3
      phosphatidylinositol 3,4,5-triphosphate
      DMEM
      Dulbecco's modified Eagle's medium
      KRB
      Kreb's Ringer buffer
      5-HT
      5-hydrotryptamine
      ROI
      regions of interest
      SERCA
      sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
      ER
      endoplasmic reticulum
      PLC
      phospholipase C
      PKC
      protein kinase C
      Insulin secreted by pancreatic β-cells is the primary regulator of serum glucose concentrations in mammals. Although substantial progress has been made in elucidating the mechanisms responsible for normal regulation of insulin secretion from the β-cell, many aspects of this process remain unclear. In particular, chemical and physiological interactions between cells within the islet exert an important level of control in the physiological regulation of insulin secretion that is not entirely understood. Both hormonal and neuronal influences within islets may modulate β-cell activity and insulin secretion in vitro and in vivo (
      • Schuit F.C.
      • Derde M.P.
      • Pipeleers D.G.
      ,
      • Norman A.W.
      • Litwack G.
      ,
      • Moens K.
      • Himberg H.
      • Flamez D.
      • Huypens P.
      • Quartier E.
      • Ling Z.
      • Pipeleers D.
      • Gremlich S.
      • Thorens B.
      • Schuit F.
      ). Although such influences have been demonstrated, the existence of significant autocrine effects of insulin on β-cells remained controversial for many years because a variety of studies yielded conflicting evidence on the modulation of insulin secretion by insulin in whole islets orin vivo. Recently, however, a variety of new methods have been utilized that demonstrate potent and possibly clinically important autocrine actions of insulin.
      Several recent studies have indicated that β-cells express components of insulin signaling systems including insulin receptors (
      • Verspohl E.J.
      • Ammon H.P.T.
      ,
      • Gazzano H.
      • Halban P.
      • Prentki M.
      • Ballotti R.
      • Brandenburg D.
      • Fehlmann M.
      • Van Obberghen E.
      ,
      • Rothenberg P.L.
      • Willison L.D.
      • Simon J.
      • Wolf B.A.
      ), insulin receptor substrates (IRS-1 and IRS-2)1 (
      • Harbeck M.C.
      • Louie D.C.
      • Howland J.
      • Wolf B.A.
      • Rothenberg P.L.
      ,
      • Velloso L.A.
      • Carneiro E.M.
      • Crepaldi S.C.
      • Boschero A.C.
      • Saad M.J.A.
      ,
      • Sun X.J.
      • Pons S.
      • Wang L.M.
      • Zhang Y.T.
      • Yenush L.
      • Burks D.
      • Myers M.G.
      • Glasheen E.
      • Copeland N.G.
      • Jenkins N.A.
      • Pierce J.H.
      • White M.F.
      ), phosphatidylinositol 3-kinase (PI3-K) (
      • Alter C.A.
      • Wolf B.A.
      ,
      • Gao Z.-Y.
      • Konrad R.J.
      • Collins H.
      • Matschinsky F.M.
      • Rothenberg P.L.
      • Wolf B.A.
      ), and protein kinase B (
      • Holst L.S.
      • Mulder H.
      • Manganiello V.
      • Sundler F.
      • Ahren B.
      • Holm C.
      • Degerman E.
      ). Evidence has also been obtained indicating that insulin released by glucose can activate these components in addition to other proteins in the cells. Insulin binds to receptors on the surface of β-cells (
      • Verspohl E.J.
      • Ammon H.P.T.
      ,
      • Patel Y.C.
      • Amherdt M.
      • Orci L.
      ) and activates tyrosine phosphorylation of insulin receptors (
      • Rothenberg P.L.
      • Willison L.D.
      • Simon J.
      • Wolf B.A.
      ), insulin receptor substrates (
      • Velloso L.A.
      • Carneiro E.M.
      • Crepaldi S.C.
      • Boschero A.C.
      • Saad M.J.A.
      ), and PHAS-I (an inhibitor of mRNA cap-binding protein) (
      • Xu G.
      • Marshall C.A.
      • Lin T.-A.
      • Kwon G.
      • Munivenkatappa R.B.
      • Hill J.R.
      • Lawrence Jr., J.C.
      • McDaniel M.L.
      ). Furthermore, maximal glucose-stimulated production of phosphatidylinositol 3,4,5-triphosphate (PIP3), a major product of PI3-K activity, coincides with the early peak phase insulin secretion in islets and clonal β-cells (
      • Alter C.A.
      • Wolf B.A.
      ). Thus, autocrine activation of the β-cell insulin receptors and several downstream proteins has been demonstrated.
      Some of the physiological consequences of insulin receptor activation at β-cells have recently been revealed. Activation of the insulin signaling pathway in β-cells leads to initiation of insulin synthesis at both transcriptional and translational levels, increasing the cellular content of releasable hormone in primary and clonal β-cell cultures (
      • Xu G.
      • Marshall C.A.
      • Lin T.-A.
      • Kwon G.
      • Munivenkatappa R.B.
      • Hill J.R.
      • Lawrence Jr., J.C.
      • McDaniel M.L.
      ,
      • Xu G.
      • Rothenberg P.L.
      ,
      • Leibiger I.B.
      • Leibiger B.
      • Moede T.
      • Berggren P.-O.
      ). In βTC6-F7 cells transfected to overexpress the insulin receptor, basal and glucose-stimulated insulin secretion was enhanced compared with kinase negative controls (
      • Xu G.
      • Rothenberg P.L.
      ). In another report, clonal cells lacking the IRS-1 protein showed both decreased insulin content and glucose-stimulated secretion (
      • Kulkarni R.N.
      • Winnay J.N.
      • Brüning J.C.
      • Hanahan D.
      ). These latter studies suggest that insulin can exert positive control over synthesis and/or secretion. Direct evidence for the effects of insulin on insulin secretion has been obtained by application of exogenous insulin to isolated β-cells and detecting secretion by amperometry (
      • Aspinwall C.A.
      • Lakey J.R.T.
      • Kennedy R.T.
      ). These data illustrate that insulin evokes insulin secretion mediated by the insulin receptor and that such positive feedback occurs during glucose stimulation. This report also showed that insulin could evoke an increase in intracellular [Ca2+] ([Ca2+]i). A recent study with clonal β-cells demonstrated that overexpression of IRS-1 and insulin receptor elevated [Ca2+]i levels and enhanced fractional insulin secretion (
      • Xu G.G.
      • Gao Z.Y.
      • Borge P.D.
      • Wolf B.A.
      ), in good agreement with the studies on application of exogenous insulin.
      The potential in vivo significance of positive autocrine feedback on insulin secretion and synthesis was revealed in experiments in which the gene for the β-cell insulin receptor was inactivated by use of the Cre-loxP system (
      • Kulkarni R.N.
      • Brüning J.C.
      • Winnay J.N.
      • Postic C.
      • Magnuson M.A.
      • Kahn C.R.
      ). Mice lacking the β-cell insulin receptor had lowered insulin response to glucose and impaired glucose tolerance, suggesting an important role for autocrine signaling in insulin secretion and glucose homeostasis in vivo. Further evidence for the importance of autocrine action was obtained when a polymorphism in IRS-1 in humans was associated with impaired insulin secretion and pathology of some forms of type 2 diabetes (
      • Porzio O.
      • Federici M.
      • Hribal M.L.
      • Lauro D.
      • Accili D.
      • Lauro R.
      • Borboni P.
      • Sesti G.
      ). The identical polymorphism expressed in clonal β-cells reduced glucose and sulfonylurea-stimulated insulin secretion.
      The evidence so far has established that insulin activates the insulin receptor and that this effect results in enhanced insulin synthesis and insulin secretion. Derangement in this process leads to impaired insulin secretion similar to that seen in type 2 diabetes. Such results suggest a potential link between the symptoms of insulin resistance and impaired insulin secretion found in type 2 diabetes. Given the potential significance of autocrine activation of insulin secretion and [Ca2+]i changes, we have investigated some of the important elements that couple an insulin stimulus to insulin secretion and [Ca2+]i changes and further characterized the source and temporal characteristics of the [Ca2+]i changes.

      DISCUSSION

      The discovery that β-cell insulin receptors play a role in normal regulation of insulin secretion provides a potential direct link between impaired insulin secretion and insulin resistance in type 2 diabetes (
      • Aspinwall C.A.
      • Lakey J.R.T.
      • Kennedy R.T.
      ,
      • Kulkarni R.N.
      • Brüning J.C.
      • Winnay J.N.
      • Postic C.
      • Magnuson M.A.
      • Kahn C.R.
      ,
      • Rutter G.A.
      , ). Investigation of the signal transduction mechanisms by which insulin exerts the stimulatory effect on insulin secretion from the β-cell is therefore essential. Our data have shown that insulin-stimulated insulin secretion is mediated by functional insulin receptors (
      • Aspinwall C.A.
      • Lakey J.R.T.
      • Kennedy R.T.
      ), IRS-1, and PI3-K activation. Activation of these substrates by insulin in β-cells has previously been reported (
      • Leibiger I.B.
      • Leibiger B.
      • Moede T.
      • Berggren P.-O.
      ,
      • Xu G.G.
      • Gao Z.Y.
      • Borge P.D.
      • Wolf B.A.
      ), and our results illustrate that these effects are directly linked to insulin secretion and increases in [Ca2+]i.
      The increase in [Ca2+]i evoked by insulin appears to be mediated by release of Ca2+ from intracellular Ca2+ stores based on the localization of the increase, the effects of thapsigargin, and the occurrence of [Ca2+]i changes in the absence of extracellular Ca2+. The release of intracellular Ca2+requires activation of IRS-1/PI3-K; however, the complete biochemical mechanism is not clear. The PLC inhibitor study indicates that Ca2+ release does not result from PIP3activation of PLC via PI3-K; however, because multiple isoforms of PLC exist and the inhibitor used may not cross-react with all isoforms (
      • Zawalich W.S.
      • Zawalich K.C.
      • Kelley G.G.
      ), it is not possible to completely rule out a role for any isoform of PLC in the insulin signaling pathway. One possible explanation of the increases in [Ca2+]i is due to the inhibition of SERCA pumps on the ER. IRS-1 has been shown to interact with SERCA proteins (
      • Algenstaedt P.
      • Antonetti D.A.
      • Yaffe M.B.
      • Kahn C.R.
      ), and it has recently been demonstrated that overexpression of IRS-1 in the clonal β-cell line βTC6-F7 inhibits the uptake of Ca2+ into ER, possibly by inhibition of the SERCA pump (
      • Xu G.G.
      • Gao Z.Y.
      • Borge P.D.
      • Wolf B.A.
      ). The slow time course of the insulin-induced [Ca2+]i changes is consistent with a mechanism involving inhibition of the SERCA pump; however, further experiments would be needed to establish this link.
      An important question is whether the increased [Ca2+]i evoked by insulin is required for the detected exocytosis. In our experiments, we found that any treatment that eliminated the [Ca2+]i increase (IRS-1 knockout, PI3-K inhibition, or thapsigargin treatment) also eliminated secretion. An important coupling point between [Ca2+]i increases and exocytosis in β-cells is PKC. PKC can be activated by Ca2+ (
      • Ashcroft S.J.
      ), and its activation can elicit exocytosis in β-cells independent of entry of Ca2+ from the extracellular environment (
      • Eliasson L
      • Renstrom E
      • Ammala C
      • Berggren P.O.
      • Bertorello A.M.
      • Bokvist K.
      • Chibalin A.
      • Deeney J.T.
      • Flatt P.R.
      • Gabel J.
      • Gromada J.
      • Larsson O.
      • Lindstrom P.
      • Rhodes C.J.
      • Rorsman P.
      ). Our data would support the hypothesis that IRS-1/PI3-K-mediated increases in [Ca2+]i are necessary for insulin-evoked exocytosis and that the [Ca2+]i changes and secretion are linked at least by PKC, if not at other points in the regulated exocytosis pathway. An interesting point in the link between insulin-stimulated [Ca2+]i changes and exocytosis is the observation that [Ca2+]i changes were generally prolonged, typically lasting more than a minute after a 30-s stimulation, but the secretory activity that we detected usually occurred during a 30-s stimulation. This differential time course suggests that other factors are necessary for secretion in the presence of elevated [Ca2+]i. Such factors would presumably be normally provided by glucose metabolism.
      We have shown that inhibition of PI3-K blocks both the [Ca2+]i increase and exocytosis evoked by insulin. PI3-K may be involved in releasing intracellular Ca2+ through an interaction with the ER, and the resulting rise in [Ca2+]i may be sufficient for activating secretion; however, we cannot rule out that PI3-K has other roles in activating exocytosis. Several lines of evidence suggest that phosphorylated products of phosphatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway (
      • De Camilli P.
      • Scott D.E.
      • McPherson P.S.
      • Novick P.
      ,
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ). PI3-K also forms an essential link between the insulin receptor and glucose transporter translocation and intracellular vesicular trafficking (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ). In addition, PI3-K has been shown to be involved in regulated exocytosis in adrenal chromaffin cells by an interaction with the actin cytoskeleton independent of any effects on Ca2+ (
      • Chasserot-Golaz S.
      • Hubert P.
      • Thierse D.
      • Dirrig S.
      • Vlahos C.J.
      • Aunis D.
      • Bader M.F.
      ). A direct link between PI3-K and the late granule docking step of regulated exocytosis was also suggested from a recent report that synaptotagmin interacts with PIP2 and PIP3 in a Ca2+-dependent manner (
      • Schiavo G., GU, Q.M.
      • Pretwich G.D.
      • Sollner T.H.
      • Rothman J.E.
      ). Thus, the involvement of PI3-K in autocrine activation of insulin secretion opens up a number of possible routes for secretion regulation in β-cells.
      Fig. 6 presents a summary of the possible pathways for the effects of insulin on Ca2+ and insulin secretion within the β-cell based on the data presented here. Autocrine activation of insulin secretion in the β-cell is mediated by activation of IRS-1 and PI3-K. PI3-K or its phosphatidylinositol products may be involved, with Ca2+, in direct activation of the exocytosis machinery of the cell. IRS-1/PI3-K also evokes release of Ca2+ from the ER by an as yet unknown mechanism. The Ca2+ may be directly involved in activating exocytosis; however, our data favor a requirement for PKC activation. Although our results have emphasized autocrine activation of an insulin receptor/IRS-1 pathway, previous investigations have demonstrated a significant role for IRS-2 activation as well. Increased insulin biosynthesis may be mediated by autocrine activation of IRS-2 (
      • Leibiger I.B.
      • Leibiger B.
      • Moede T.
      • Berggren P.-O.
      ). In addition, mice with IRS-2 gene knockouts show defects in islet development (
      • Withers D.J.
      • Gutierrez J.S.
      • Towery H.
      • Burks D.J.
      • Ren J.-M.
      • Previs S.
      • Zhang Y.
      • Bernal D.
      • Pons S.
      • Shulman G.I.
      • Bonner-Weir S.
      • White M.F.
      ) and IRS-2 in β-cells may mediate IGF-1 receptor effects on β-cell development and peripheral insulin signaling (
      • Withers D.J.
      • Burks D.J.
      • Towery H.
      • Altamuro S.L.
      • Flint C.L.
      • White M.F.
      ).
      Figure thumbnail gr6
      Figure 6Summary of insulin signaling pathway involved in autocrine activation of secretion in pancreatic β-cell. Solid lines indicate established effects or interactions, whereas dashed lines indicate events with an unclear mechanism. Insulin applied exogenously or released from secretory granules (SG) binds the insulin receptor (IR), which activates IRS-1/PI3-K leading to release of intracellular Ca2+ from the ER by an unknown mechanism. Increased [Ca2+]i leads to insulin secretion mediated by PKC. The possibility also exists for a more direct involvement of PI3-K in activating exocytosis.
      Our data have identified some important contributors to the observed activation of insulin secretion and increased [Ca2+]i evoked by insulin at the β-cell. These mechanisms are presumably activated by insulin released during normal glucose stimulation in vivo. The importance of these effects for normal glucose homeostasis has been demonstrated by the glucose intolerance and reduction of first phase glucose-stimulated insulin secretion in mice lacking the β-cell insulin receptor (
      • Kulkarni R.N.
      • Brüning J.C.
      • Winnay J.N.
      • Postic C.
      • Magnuson M.A.
      • Kahn C.R.
      ) and impaired insulin secretion associated with IRS-1 polymorphisms (
      • Porzio O.
      • Federici M.
      • Hribal M.L.
      • Lauro D.
      • Accili D.
      • Lauro R.
      • Borboni P.
      • Sesti G.
      ). Further studies are needed to understand the linkage between effects regulated by glucose versus insulin and possible interactions of insulin with metabolism in the β-cell. Defects in any of the components of the insulin signaling pathway could be involved in impaired insulin secretion and insulin resistance seen in diabetes; however, the actual role of autocrine regulation in diabetes remains to be determined.

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