Insulin induces calcium signals in the nucleus of rat hepatocytes

Abstract

Insulin is an hepatic mitogen that promotes liver regeneration. Actions of insulin are mediated by the insulin receptor, which is a receptor tyrosine kinase. It is currently thought that signaling via the insulin receptor occurs at the plasma membrane, where it binds to insulin. Here we report that insulin induces calcium oscillations in isolated rat hepatocytes, and that these calcium signals depend upon activation of phospholipase C and the inositol 1,4,5-trisphosphate receptor, but not upon extracellular calcium. Furthermore, insulin-induced calcium signals occur in the nucleus, and are temporally associated with selective depletion of nuclear phosphatidylinositol bisphosphate and translocation of the insulin receptor to the nucleus. These findings suggest that the insulin receptor translocates to the nucleus to initiate nuclear, inositol 1,4,5-trisphosphate-mediated calcium signals in rat hepatocytes. This novel signaling mechanism may be responsible for insulin's effects on liver growth and regeneration. ( I nsulin regulates a wide variety of biological functions in the liver, including glucose uptake, 1 regulation of gene expression, 2 and promotion of cell growth. [3][4][5] The biological actions of insulin are initiated by binding to the insulin receptor, a heterotetrameric receptor tyrosine kinase (RTK) composed of two extracellular ␣-subunits and two transmembrane ␤-subunits. 6 The ␣-subunit possesses insulin-binding activity whereas the ␤-subunit has intrinsic protein tyrosine kinase activity. Binding of insulin to the ␣-subunit of its receptor activates the protein tyrosine kinase and results in phosphorylation of tyrosine residues of the ␤-subunit and of several endogenous substrates. These substrates include proteins containing a src-homology 2 domain such as phosphatidylinositol 3-kinase and phospholipase C (PLC). 7 PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ), generating two intracellular products: inositol 1,4,5-trisphosphate (InsP3), a universal calcium-mobilizing second messenger, and diacylglycerol, an activator of protein kinase C. Like insulin, Ca 2ϩ also regulates glucose metabolism, 8 gene expression, 9,10 and cell growth. 11,12 Although it has not been established how a single second messenger coordinates such diverse effects within a cell, there is increasing evidence that the spatial and temporal patterns of Ca 2ϩ signals may determine their specificity. Ca 2ϩ signaling patterns can vary in different regions of the cell, and increases in Ca 2ϩ in the nucleus have specific biological effects that differ from the effects of increases in cytosolic Ca 2ϩ . 9,10,[13][14][15] The mechanisms and pathways that promote localized increases in free Ca 2ϩ levels in the nucleus have not been entirely defined. It is currently thought that signaling via the insulin receptor occurs only at the plasma membrane, where it binds to insulin. 16 Here we investigate whether and how insulin signaling occurs in the nucleus of hepatocytes, where its downstream messenger Ca 2ϩ may act. Materials...

Nuclear inositol 1,4,5-trisphosphate is a necessary and conserved signal for the induction of both pathological and physiological cardiomyocyte hypertrophy

“…The components include PIP kinase (PIPK), which synthesizes PIP 2 , PLC, as well as functional G-protein couple receptors [37][38][39][40], and tyrosine kinase receptors [14,22,41], that once activated are known to trigger nuclear PIP 2 hydrolysis, generating IP3 in the nucleoplasm [14,22]. Additionally, IP3R is known to be expressed in the nucleus of several cell types [42,43].…”

“…The ability of IP3 to bind to the IP3 binding region of the type I IP3R construct had already been demonstrated [21]. Moreover, we had previously shown the efficiency and selectivity of the IP3 vectors to buffer agonist triggering IP3 dependent Ca 2+ signals, either in the nucleus or in the cytosol [14,22]. In the current work, we expressed the targeted IP3 buffer constructs and a control vector in primary culture of neonatal cardiomyocytes Pink represent images of nucleus staining with Dapi (blue) merged with IP3-buffer expressing in the nucleus (red).…”

PIP5K-driven PtdIns(4,5)P2 synthesis: regulation and cellular functions

“…Receptors for Ins(1,4,5)P 3 are present in the inner nuclear envelope and application of Ins(1,4,5)P 3 to isolated nuclei induces the release of Ca 2+ from the nuclear envelope into the nucleus (Malviya et al, 1990;Matter et al, 1993). Recently, agonist-stimulated changes in nuclear Ca 2+ acting downstream of a nuclear PLC pathway have been convincingly demonstrated (Kumar et al, 2008;Rodrigues et al, 2008). Ins(1,4,5)P 3 can also be further phosphorylated to higher inositol phosphates that, in yeast, have functions in regulating mRNA export (York et al, 1999) and gene transcription (Odom et al, 2000;Jones and Divecha, 2004).…”

Insulin stimulates fibroblast proliferation through calcium-calmodulin-dependent kinase II

“…This enzyme is activated by insulin stimulation of the IR and it has recently been shown to participate to insulin stimulated proliferation in 3T3-L1 adipocytes, Rat-1 fibroblasts and hepatocytes. [12][13][14] An important mediator of Ca 2+ signal is the calcium calmodulin-dependent kinase II (CaMKII), a ubiquitous serine-threonine kinase that phosphorylates a large number of substrates. 15,16 There is an extensive cross-talk between CaMKs and other signaling cascades, including those that involve the cAMP-dependent kinase (PKA), ERK and Akt/PKB.…”