Insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades involving GSK-3beta inhibition and Ras activation.

We examined the interplay between the insulin/IGF-1- and beta-catenin-regulated pathways, both of which are suspected to play a role in hepatocarcinogenesis. Insulin and IGF-1 stimulated the transcription of a Lef/Tcf-dependent luciferase reporter gene by 3-4-fold in HepG2 cells. This stimulation was mediated through the activation of phosphatidylinositol 3-kinase (PI 3-K)/Akt and the inhibition of glycogen synthase kinase-3beta (GSK-3beta) since the effects of insulin and IGF-1 were inhibited by dominant-negative mutants of PI 3-K or Akt and an uninhibitable GSK-3beta. Together with inhibiting GSK-3beta, insulin and IGF-1 increased the cytoplasmic levels of beta-catenin. The PI 3-K/Akt/GSK-3beta pathway was not the sole to mediate insulin and IGF-1 stimulation of Lef/Tcf-dependent transcription. The Ras signalling pathway was also required as (i) the stimulatory effects of insulin and IGF-1 were inhibited by dominant-negative Ras or the MEK1 inhibitor PD98059 and (ii) activated Ha-Ras or constitutively active MEK1 synergized with catalytically inactive GSK-3beta to stimulate Lef/Tcf-dependent transcription. This study provides the first evidence that insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades bifurcating downstream of PI 3-K and involving GSK-3beta inhibition and Ras activation. These findings demonstrate for the first time the ability of insulin and IGF-1 to activate the beta-catenin pathway in hepatoma cells and thereby provide new insights into the role of these factors in hepatocarcinogenesis.

Insulin-mediated cell proliferation and survival involve inhibition of c-Jun N-terminal kinases through a phosphatidylinositol 3-kinase- and mitogen-activated protein kinase phosphatase-1-dependent pathway.

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.

Lipoatrophic diabetes: genetic exclusion of the insulin receptor gene.

Lipoatrophic diabetes (LD) is a syndrome with congenital or delayed onset, characterized by severe insulin resistance and generalized lipoatrophy. Using denaturing gradient gel electrophoresis and sequencing, we have investigated the contribution of defects in the insulin receptor (IR) gene in LD. First, we performed an association study between the IR gene and congenital lipoatrophy in two families with consanguineous parents and one or two affected children (patients D1, D2, and D3). Segregation analysis of intragenic polymorphisms excluded a linkage between the IR locus and the LD phenotype in both families. Second, we screened for mutations in all exons and splice site junctions of the IR gene from patients D1-D3 and 11 additional unrelated patients with congenital or delayed forms of LD. The IR sequence proved to be normal in all 14 subjects because nucleotide variations that we detected were silent. The relative levels of expression of the 2 alleles of the IR gene were evaluated by allele-specific oligonucleotide hybridization in cells from most of these patients, and no gross alteration was detected. Overall, these results provide the first clear evidence against the involvement of the IR gene in the pathogenesis of any clinical form of LD.

Antiinsulin receptor autoantibodies induce insulin receptors to constitutively associate with insulin receptor substrate-1 and -2 and cause severe cell resistance to both insulin and insulin-like growth factor I.

We report here that antiinsulin receptor (anti-IR) autoantibodies (AIRs) from a newly diagnosed patient with type B syndrome of insulin resistance induced cellular resistance not only to insulin but also to insulin-like growth factor I (IGF-I) for the stimulation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase activities and of glycogen and DNA syntheses. The molecular mechanisms of this dual resistance were investigated. Patient AIRs bound the IR at the insulin-binding site and caused insulin resistance at the IR level by inducing a 50% decrease in cell surface IRs and a severe defect in the tyrosine kinase activity of the residual IRs, manifested by a loss of insulin-stimulated IR autophosphorylation and IR substrate-1 (IRS-1)/IRS-2 phosphorylation. In contrast, cell resistance to IGF-I occurred at a step distal to IGF-I receptors (IGF-IRs), as AIRs altered neither IGF-I binding nor IGF-I-induced IGF-IR autophosphorylation, but inhibited the ability of IGF-IRs to mediate tyrosine phosphorylation of IRS-1 and IRS-2 in response to IGF-I. Coimmunoprecipitation assays showed that in AIR-treated cells, IRs, but not IGF-IRs, were constitutively associated with IRS-1 and IRS-2, strongly suggesting that AIR-desensitized IRs impeded IGF-I action by sequestering IRS-1 and IRS-2. Accordingly, AIRs had no effect on the stimulation of mitogen-activated protein kinase activity or DNA synthesis by vanadyl sulfate, FCS, epidermal growth factor, or platelet-derived growth factor, all of which activate signaling pathways independent of IRS-1/IRS-2. Thus, AIRs induced cell resistance to both insulin and IGF-I through a novel mechanism involving a constitutive and stable association of IRS-1 and IRS-2 with the IR.

Deletion of Asn281 in the alpha-subunit of the human insulin receptor causes constitutive activation of the receptor and insulin desensitization.

We studied the structure and function of the insulin receptor (IR) in two sisters with leprechaunism. The patients had inherited alterations in the IR gene and were compound heterozygotes. Their paternal IR allele carried a major deletion, including exons 10-13, which shifted the reading frame and introduced a premature chain termination codon in the IR sequence. This allele was expressed at a very low level in cultured fibroblasts (< 10% of total IR messenger ribonucleic acid content) and encoded a truncated protein lacking transmembrane and tyrosine kinase domains. The maternal IR allele was deleted of 3 bp in exon 3, causing the loss of Asn281 in the alpha-subunit. This allele generated levels of IR messenger ribonucleic acid and cell surface receptors similar to those seen in control fibroblasts. However, IRs from patients' cells had impaired insulin binding and exhibited in vivo and in vitro constitutive activation of autophosphorylation and tyrosine kinase activity. As a result of this IR-preactivated state, the cells were desensitized to insulin stimulation of glycogen and DNA syntheses. These findings strongly suggest that Asn281 of the IR alpha-subunit plays a critical role in the inhibitory constraint exerted by the extracellular alpha-subunit over the intracellular kinase activity.

Severe resistance to insulin and insulin-like growth factor-I in cells from a patient with leprechaunism as a result of two mutations in the tyrosine kinase domain of the insulin receptor.

We studied the biological properties of insulin receptors (IRs) and insulin-like growth factor-I (IGF-I) receptors in cultured fibroblasts from a patient with leprechaunism (leprechaun Par-1). Patient cells displayed normal insulin binding capacity and affinity. Basal in vivo autophosphorylation and in vitro exogenous kinase activity of patient IRs were elevated twofold to threefold compared with control receptors, and insulin had no further effect on these processes. Moreover, patient IRs were unable to promote the stimulation of metabolic and mitogenic pathways. IR substrate-1 (IRS-1) and mitogen-activated protein (MAP) kinase tyrosine phosphorylation and glycogen and DNA synthesis were not increased in the basal state in patient fibroblasts and were also insensitive to the stimulatory effect of insulin. As for IGF-I, although binding and receptor kinase activity were normal, the ability to stimulate glycogen and DNA synthesis was altered in patient cells. Two mutant alleles of the IR gene were detected by denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The maternal allele contained a point mutation in exon 18 encoding the tryptophan-for-arginine substitution at position 1092, and the paternal allele had a point mutation in exon 20 substituting lysine for glutamic acid at codon 1179. Thereby, leprechaun Par-1 was a compound heterozygote for two missense mutations located in the IR beta-subunit. The present investigation provides the first evidence that leprechaunism can be causally related to structural alterations in the tyrosine kinase domain of the IR. These alterations result in severe impairment of insulin and IGF-I action.

Involvement of small ArfGAP1 (SMAP1), a novel Arf6-specific GTPase-activating protein, in microsatellite instability oncogenesis.

Small ArfGAP1 (stromal membrane-associated protein 1, SMAP1), a GTPase-activating protein specific for ADP-ribosylation factor 6 (Arf6), which is a small GTPase acting on membrane trafficking and actin remodeling, is frequently mutated in various tumors displaying microsatellite instability (MSI), notably in MSI colorectal cancers (CRC). Genotyping of 93 MSI CRCs (40 stage II, 32 stage III and 21 stage IV) allowed us to underscore that SMAP1 mutation frequency was inversely correlated with disease stage (P=0.01). Analysis of 46 cancer cell lines showed that SMAP1 mutations occurred only in MSI tumors, and consisted exclusively in short insertion or deletion in the coding 10-adenine repeat, generating a premature termination codon located downstream the ArfGAP domain. SMAP1 transcript levels were significant decreased (P=0.006), and truncated SMAP1 protein could not be detected in cells displaying biallelic SMAP1 mutations, owing to its sensitivity to proteasome degradation. To investigate the role of SMAP1 mutations, we used the SMAP1-null HCT116 cell line and we established three isogenic SMAP1-complemented clones. Cell proliferation was first assessed in vivo using subcutaneous xenografts into immunodeficient mice. Tumors developed in all animals regardless of the cell line injected, but tumor volumes were significantly smaller for both SMAP1-complemented clones compared with HCT116 (P<0.0001, at the time of killing). In vitro, SMAP1 mutations also increased cell clonogenicity (P=0.02-0.04), cell proliferation (P=0.008) by shortening the G2/M phase and decreased cell invasiveness (P=0.03-0.003). In keeping, SMAP1-complemented HCT116 gained several mesenchymal markers (Snail, Slug and vimentin) considered as a hallmark of epithelial-to-mesenchymal transition. These observations are reminiscent of some clinical characteristics of MSI CRCs, notably their larger size and lower rate of metastasis. Our observations suggest that SMAP1 loss-of-function mutations in MSI CRC may contribute to the emerging oncogenic pathway involving abnormal Arf6 regulation.

Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells.

Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis through EGFR activation.

Insulin differentially regulates SAPKs/JNKs and ERKs in CHO cells overexpressing human insulin receptors.

In the present study, we compared the ability of insulin to regulate SAPKs/JNKs and ERKs in CHO cells overexpressing human insulin receptors. We show that acute insulin treatment induced a time-dependent increase both in SAPK/JNK and ERK activity but with distinct kinetics. PI-3-kinase inhibition by wortmannin completely blocked insulin activation of SAPKs/JNKs, whereas it partially decreased ERK activation. Prolonged exposure to insulin caused a marked inhibition of SAPK/JNK activity while it induced a sustained activation of ERKs. Insulin inhibition of SAPKs/JNKs was partly due to decreased tyrosine phosphorylation of JNK2. These data indicate that insulin differentially regulates SAPKs/JNKs and ERKs. Moreover, they provide the first evidence that insulin exerts opposite effects on SAPK/JNK activity according to the time of cell treatment.

Insulin antiapoptotic signaling involves insulin activation of the nuclear factor kappaB-dependent survival genes encoding tumor necrosis factor receptor-associated factor 2 and manganese-superoxide dismutase.

We recently showed that the antiapoptotic function of insulin requires nuclear factor kappaB (NF-kappaB) activation (Bertrand, F., Atfi, A., Cadoret, A., L'Allemain, G., Robin, H., Lascols, O., Capeau, J., and Cherqui, G. (1998) J. Biol. Chem. 273, 2931-2938). Here we sought to identify the NF-kappaB-dependent survival genes that are activated by insulin to mediate this function. Insulin increased the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2) mRNA and protein in Chinese hamster ovary cells overexpressing insulin receptors (IRs). This effect required (i) IR activation since it was abrogated by IR mutation at tyrosines 1162 and 1163 and (ii) NF-kappaB activation since it was abolished by overexpression of dominant-negative IkappaB-alpha(A32/36) and mimicked by overexpression of the NF-kappaB c-Rel subunit. TRAF2 contributed to insulin protection against serum withdrawal-induced apoptosis since TRAF2 overexpression mimicked insulin protection, whereas overexpression of dominant-negative TRAF2-(87-501) reduced this process. Along with its protective effect, overexpressed TRAF2 increased basal and insulin-stimulated NF-kappaB activities. All effects were inhibited by IkappaB-alpha(A32/36), suggesting that an amplification loop involving TRAF2 activation of NF-kappaB is implicated in insulin antiapoptotic signaling. We also show that insulin increased manganese-superoxide dismutase (Mn-SOD) mRNA expression through NF-kappaB activation and that Mn-SOD contributed to insulin antiapoptotic signaling since expression of antisense Mn-SOD RNA decreased this process. This study provides the first evidence that insulin activates the NF-kappaB-dependent survival genes encoding TRAF2 and Mn-SOD and thereby clarifies the role of NF-kappaB in the antiapoptotic function of insulin.

[Major insulin resistance syndromes: clinical and physiopathological aspects]. / Syndromes d'insulino-resistance majeure: clinique et physiopathologie.

Insulin resistance is a common metabolic disorder. It plays an important role in the metabolic syndrome (or syndrome X), type 2 diabetes, obesity and in the lipodystrophic syndromes recently described, associated with treatments of HIV disease and represent a worrying cardiovascular risk. However, its pathophysiology remains poorly understood in these situations. Syndromes of major insulin resistance, although rare, allow investigations of the mechanisms leading to alterations in the insulin transduction pathways. Mutations of the insulin receptor gene have been discovered in rare patients. Therefore alterations at the post-receptor level are probably causative in other cases. Furthermore, the role of body fat repartition seems determinant in the apparition of insulin resistance, as attested by the clinical characteristics of lipodystrophies, either congenital or acquired. The two lipodystrophic syndromes which molecular defect is identified are the familial partial lipodystrophy of the Dunnigan type, due to mutations of the lamin A/C gene, and the congenital generalized lipodystrophy, linked to alterations in the protein seipin. However, their physiopathology remains mysterious. Lamin A/C is indeed an ubiquitous nuclear protein, which is also mutated in a genetic squelettic and/or cardiac myopathy, and seipin is a protein of unknown function mainly expressed in brain. Progresses in the understanding of these syndromes, in particular lipodystrophies which can be considered as caricatural models of the metabolic syndrome, will probably allow to clarify the physiopathology of the more common forms of insulin resistance.

Molecular analysis of the insulin receptor gene for prenatal diagnosis of leprechaunism in two families.

Leprechaunism is a rare autosomal recessive disorder characterized by marked intrauterine and postnatal growth retardation, severe insulin resistance, and altered glucose homeostasis. This syndrome is related to mutations in the insulin receptor (IR) gene that impair the transmission of the insulin signal by several mechanisms. There is no effective therapy and patients usually die within the first months of life. Here we report the prenatal diagnosis of leprechaunism in two unrelated families in which affected children were compound heterozygotes with two different deficient IR alleles. In family Par-1, the disease IR alleles carried a missense mutation located in exon 18 (Arg1092-->Trp) and exon 20 (Glu1179-->Lys). In family Als, a 3-basepair deletion causing the loss of Asn281 in exon 3 and a major deletion of exons 10-13 were present in the maternal and paternal mutant IR alleles, respectively. Prenatal diagnosis was made in each family by a specific approach combining denaturing gradient gel electrophoresis (DGGE) and Southern blotting. This methodology allowed us to correctly predict the genotype of the two fetuses at the IR locus.

Major circadian variations of glucose homeostasis in a patient with Rabson-Mendenhall syndrome and primary insulin resistance due to a mutation (Cys284-->Tyr) in the insulin receptor alpha-subunit.

We have performed clinical, in vitro biochemical, and genetic studies of a patient with severe insulin resistance, considerable growth restriction, and Rabson-Mendenhall syndrome (patient RM-3). The blood IGF-I level was undetectable in this patient, although the GH level was moderately decreased. During the postprandial period, glycemia, ketonuria, and plasma glucagon were very elevated despite high doses of exogenous insulin (glucose levels up to 30 mmol/L). In the postabsorptive state, blood glucose was normalized with small amounts of insulin; ketonuria, and glucagon levels were reduced but remained supranormal. Erythrocytes and cultured skin fibroblasts from the patient displayed a decrease in cell surface insulin receptors (IRs). The ability of physiologic concentrations of insulin to stimulate metabolic processes was altered in patient fibroblasts. Analysis of the IR gene by denaturing gradient gel electrophoresis and direct sequencing showed a homozygous missense mutation in exon 3, replacing Cys284 by Tyr in the alpha-subunit. In conclusion, marked primary insulin resistance was evidenced in patient cells as a result of a structural alteration in the IR alpha-subunit. The in vitro studies could not account alone for the in vivo metabolic alterations because glucose homeostasis varied considerably during the day in the patient.