Vanadate inhibits transcription of the rat insulin receptor gene via a proximal sequence of the 5'flanking region.

Vanadate, a protein tyrosine phosphatase inhibitor which elicits insulin-like effects, has previously been shown to inhibit expression of the insulin receptor gene at the transcriptional level in rat hepatoma cells. In an attempt to identify the DNA sequence and transcription factors potentially involved in this effect, a fragment of the proximal 5'flanking region of the IR gene (-1143/-252 upstream the ATG codon) has been cloned and functionally characterized. RNase protection allowed the identification of several transcription start sites in the conserved region of the gene, among which two major sites at -455 and -396. Upon fusion to the luciferase gene and transient transfection into hepatoma cells, the -1143/-252 fragment showed promoter activity. This was unaffected by deletion of the -1143/-761 sequence, but markedly decreased (90%) by additional deletion of the -760/-465 sequence. Treatment of hepatoma cells with vanadate led to a dose-dependent decrease in promoter activity of the 1143/-252, -760/-252 and -464/-252 constructs (change relative to untreated cells, 40, 55 and 23% at 125 µM, and 70, 85 and 62% at 250 µM, respectively). These data suggest that although the entire DNA sequence upstream the transcription start sites is probably involved in vanadate-induced inhibition, the short sequence downstream of position -464 and is sufficient for inhibition. Potential targets of vanadate are the transcription factors FoxO1 and HMGA1, two downstream targets of the insulin signaling pathway which have been shown to mediate the inhibitory effect of insulin on IR gene expression.

Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B-chain.

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome-associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5-90 min) and was later (15-90 min) partially translocated to cytosol as A- and B-chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell-free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time-dependent translocation of ricin across the endosomal membrane occurred in cell-free endosomes. Endosome-located thioredoxin reductase-1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase-9 and caspase-3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B-chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B-chain with mitochondria is involved in ricin-induced apoptosis.

[Control of insulin signalisation and action by the Grb14 protein]. / Contrôle de la signalisation et de l'action de l'insuline par la protéine Grb14.

The action of insulin on metabolism and cell growth is mediated by a specific receptor tyrosine kinase, which, through phosphorylation of several substrates, triggers the activation of two major signaling pathways, the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the Ras/extracellular signal-regulated kinase (ERK) pathway. Insulin-induced activation of the receptor and downstream signaling is also subjected to a negative feedback control involving several mechanisms, among which the interaction of the insulin receptor and its substrates with inhibitory proteins. After summarizing the major mechanisms underlying the activation and attenuation of insulin signaling, this review focuses on its control by the Grb14 adaptor protein. Grb14 has been identif-ied as an inhibitor of insulin signaling and action, and is involved in insulin resistance associated with type 2 diabetes and obesity. Studies on the molecular mechanism of action of Grb14 have shown that, through interaction with the activated insulin receptor, Grb14 inhibits its catalytic activity and the activation of downstream signaling. However, the consequences of Grb14 gene invalidation are complex and tissue-specific, and some effects of Grb14 on insulin signaling appear to be linked to its interaction with effector proteins downstream the insulin receptor. Pharmacological inhibition of Grb14 should allow to enhance insulin sensitivity and improve energy homeostasis in insulin-resistant states.

[Involvement of the endosomal compartment in cellular insulin signaling]. / Implication du compartiment endosomal dans la signalisation cellulaire de l'insuline.

The insulin receptor and insulin signaling proteins downstream the receptor reside in different subcellular compartments and undergo redistribution within the cell upon insulin activation. Endocytosis of the insulin-receptor complex, by mediating ligand degradation and receptor dephosphorylation, is generally viewed as a mechanism which attenuates or arrests insulin signal transduction. However, several observations suggest that insulin receptor endocytosis and/or recruitement of insulin signaling proteins to endosomes are also involved in a positive regulation of insulin signaling: (1) upon internalization, the insulin receptor remains transiently phosphorylated and activated; (2) in insulin-stimulated cells or tissues, signaling proteins of the PI3K/Akt and Ras/Raf/Mek/Erk pathways are recruited to endosomes or other intracellular compartments, in which they undergo phosphorylation and/or activation; and (3) depletion or overexpression of proteins involved in the regulation of membrane trafficking and endocytosis interfere with insulin signaling. These observations support a spatial and temporal regulation of insulin signal transduction and reinforce the concept that, as for other membrane signaling receptors, endocytosis and signaling are functionally linked.

Assessment of insulin proteolysis in rat liver endosomes: its relationship to intracellular insulin signaling.

Insulin binding to insulin receptor (IR) at the cell surface results in the activation of IR kinase and initiates the translocation of insulin-IR complexes to clathrin-coated pits and to early endosomes containing internalized but still active receptors. In liver parenchyma, several mechanisms are involved in the regulation of endosomal IR tyrosine kinase activity. Two of these regulatory mechanisms are at the level of intraendosomal ligand. First, a progressive decrease in endosomal pH mediated by the vacuolar H(+)-ATPase proton pump promotes dissociation of the insulin-IR complex. Second, free dissociated insulin is degraded by a soluble endosomal acidic insulinase, which has been identified as aspartic acid protease cathepsin D. This enzyme catalyzes the cleavage of insulin at the Phe(B24)-Phe(B25) bond, generating a major clipped molecule, A(1-21)-B(1-24) insulin, that can no longer bind to IR within endosomes. Concomitant with, or shortly after, the tyrosine-phosphorylated IR is deactivated by two independent processes: its rapid dephosphorylation by endosome-associated phosphotyrosine phosphatase(s) and its association with the molecular adaptor Grb14, with resulting inhibition of IR catalytic activity. By mediating the removal and degradation of circulating insulin, as well as the deactivation of the activated IR, internalization of the insulin-receptor complex into endosomes represents a major mechanism involved in the negative regulation of insulin signaling.

Regulation of insulin and type 1 insulin-like growth factor signaling and action by the Grb10/14 and SH2B1/B2 adaptor proteins.

The effects of insulin and type 1 insulin-like growth factor (IGF-1) on metabolism, growth and survival are mediated by their association with specific receptor tyrosine kinases, which results in both receptor and substrate phosphorylation. Phosphotyrosine residues on receptors and substrates provide docking sites for signaling proteins containing SH2 (Src homology 2) domains, including molecular adaptors. This review focuses on the regulation of insulin/IGF-1 signaling and action by two adaptor families with a similar domain organization: the growth factor receptor-bound proteins Grb7/10/14 and the SH2B proteins. Both Grb10/14 and SH2B1/B2 associate with the activation loop of insulin/IGF-1 receptors through their SH2 domains, but association of Grb10/14 also involves their unique BPS domain. Consistent with Grb14 binding as a pseudosubstrate to the kinase active site, insulin/IGF-induced activation of receptors and downstream signaling pathways in cultured cells is inhibited by Grb10/14 adaptors, but is potentiated by SH2B1/B2 adaptors. Accordingly, Grb10 and Grb14 knockout mice show improved insulin/IGF sensitivity in vivo, and, for Grb10, overgrowth and increased skeketal muscle and pancreatic ß-cell mass. Conversely, SH2B1-depleted mice display insulin and IGF-1 resistance, with peripheral depletion leading to reduced adiposity and neuronal depletion leading to obesity through associated leptin resistance. Grb10/14 and SH2B1 adaptors also modulate insulin/IGF-1 action by interacting with signaling components downstream of receptors and exert several tissue-specific effects. The identification of Grb10/14 and SH2B1 as physiological regulators of insulin signaling and action, together with observations that variants at their gene loci are associated with obesity and/or insulin resistance, highlight them as potential therapeutic targets for these conditions.

Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver.

The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity. In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi/endosome fractions. Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi/endosome fraction, which paralleled the increase in IR beta-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi/endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR beta-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14-IR complex in endosomes. Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling.

Glucagon-mediated internalization of serine-phosphorylated glucagon receptor and Gsalpha in rat liver.

To assess glucagon receptor compartmentalization and signal transduction in liver parenchyma, we have studied the functional relationship between glucagon receptor endocytosis, phosphorylation and coupling to the adenylate cyclase system. Following administration of a saturating dose of glucagon to rats, a rapid internalization of glucagon receptor was observed coincident with its serine phosphorylation both at the plasma membrane and within endosomes. Co-incident with glucagon receptor endocytosis, a massive internalization of both the 45- and 47-kDa Gsalpha proteins was also observed. In contrast, no change in the subcellular distribution of adenylate cyclase or beta-arrestin 1 and 2 was observed. In response to des-His(1)-[Glu(9)]glucagon amide, a glucagon receptor antagonist, the extent and rate of glucagon receptor endocytosis and Gsalpha shift were markedly reduced compared with wild-type glucagon. However, while the glucagon analog exhibited a wild-type affinity for endosomal acidic glucagonase activity and was processed at low pH with similar kinetics and rates, its proteolysis at neutral pH was 3-fold lower. In response to tetraiodoglucagon, a glucagon receptor agonist of enhanced biological potency, glucagon receptor endocytosis and Gsalpha shift were of higher magnitude and of longer duration, and a marked and prolonged activation of adenylate cyclase both at the plasma membrane and in endosomes was observed. The subsequent post-endosomal fate of internalized Gsalpha was evaluated in a cell-free rat liver endosome-lysosome fusion system following glucagon injection. A sustained endo-lysosomal transfer of the two 45- and 47-kDa Gsalpha isoforms was observed. Therefore, these results reveal that within hepatic target cells and consequent to glucagon-mediated internalization of the serine-phosphorylated glucagon receptor and the Gsalpha protein, extended signal transduction may occur in vivo at the locus of the endo-lysosomal apparatus.

Cell itinerary and metabolic fate of proinsulin in rat liver: in vivo and in vitro studies.

Proinsulin, the insulin precursor in pancreatic beta-cells, displays a slower hepatic clearance than insulin and exerts a more prolonged metabolic effect on liver in vivo. To elucidate the mechanisms underlying these differences, the cellular itinerary and processing of proinsulin and insulin in rat liver have been comparatively studied using cell fractionation. As [125I]-insulin, [125I]-proinsulin taken up into liver in vivo was internalized and accumulated in endosomes, in which it underwent dissociation from the insulin receptor and degradation in a pH- and ATP-dependent manner. However, relative to [125I]-insulin, [125I]-proinsulin showed a delayed and prolonged in vivo association with endosomes, a slower in vivo and cell-free endosomal processing, and a higher cell-free endosome-lysosome transfer. Endosomal extracts degraded to a lesser extent proinsulin than insulin at acidic pH; so did, and even proportionally less, at neutral pH, plasma membrane and cytosolic fractions. Proinsulin degradation products generated by soluble endosomal extracts were isolated by HPLC and characterized by mass spectrometry. Under conditions resulting in multiple cleavages in insulin, proinsulin was cleaved at eight bonds in the C peptide but only at the Phe24-Phe25 bond in the insulin moiety. As native insulin, native proinsulin induced a dose- and time-dependent endocytosis and tyrosine phosphorylation of the insulin receptor; but at an inframaximal dose, proinsulin effects on these processes were of longer duration. We conclude that a reduced proteolysis of proinsulin in endosomes, and probably also at the plasma membrane, accounts for its slower hepatic clearance and prolonged effects on insulin receptor endocytosis and tyrosine phosphorylation.

The hepatocyte is a direct target for transforming-growth factor beta activation via the insulin-like growth factor II/mannose 6-phosphate receptor.

BACKGROUND/AIMS: The cation-independent mannose 6-phosphate receptor (CIMPR) is overexpressed in hepatocytes during liver regeneration and has been implicated in the maturation of latent pro-transforming growth factor beta (TGFbeta). In this study, we have: (1) kinetically characterized the changes in CIMPR expression in regenerating liver and cultured proliferating hepatocytes; and (2) assessed the contribution of hepatocyte via the CIMPR to latent pro-TGFbeta activation. METHODS: The expression of CIMPR protein and mRNA in livers collected after partial hepatectomy and hepatocyte primary cultures was analyzed by Western and Northern blotting. Activity of latent pro-TGFbeta was assessed by inhibition of [3H] methylthymidine incorporation into DNA. RESULTS: The expression of the CIMPR protein and/or mRNA progressively increased after 8 h in regenerating liver and 42-46 h in cultured hepatocytes, prior to the onset of DNA replication. Both mature TGFbeta and latent pro-TGFbeta inhibited epidermal growth factor-stimulated DNA synthesis in hepatocytes in a dose-dependent manner. The effect of latent pro-TGFbeta was reversed by two ligands of the CIMPR: beta-galactosidase, a mannose 6-phosphate containing protein, and a CIMPR antibody. CONCLUSIONS: (1) The induction of the CIMPR gene during liver regeneration and hepatocyte culture occurs in mid G1 phase; and (2) the CIMPR mediates latent proTGFbeta activation and thus may act, by targeting TGFbeta to hepatocytes, as a negative regulator of hepatocyte growth.