Gérard Crémel

Peptide-based interference of the transmembrane domain of neuropilin-1 inhibits glioma growth in vivo.

Angiogenesis in glioblastoma is largely dependent on vascular endothelial growth factor (VEGF) signalling. Consistently, the VEGF coreceptor NRP1 promotes angiogenesis and tumour growth in gliomas. Here, we provide data showing that an innovative peptidic tool targeting the transmembrane domain of NRP1 efficiently blocks rat and human glioma growth in vivo. We show both in vivo and in vitro that the antitumour effect results from the anti-proliferative, anti-migratory and anti-angiogenic properties of the compound. The proposed NRP1 antagonizing peptide is therefore a promising novel class of anti-angiogenic drugs that might prolong glioma patient survival. Our results finally show for the first time that the transmembrane domain of important signalling receptors can be antagonized in vivo thereby providing a new avenue towards the development of atypical antagonists with strong therapeutic potential.

Influence of lipid environment on insulin binding in cultured hepatoma cells

The influence of alterations of plasma membrane physico-chemical properties on insulin binding have been characterized in an insulin-sensitive rat hepatoma cell line adapted to grow for several generations in culture medium enriched with linoleic acid (18:2) or with 25-hydroxycholesterol. The cells took up 18:2 and 25-hydroxycholesterol added to the culture medium, without exhibiting any sign of intolerance or intoxication. These compounds respectively increased and decreased membrane fluidity at 37 degrees C. The cells demonstrated extensive changes in insulin binding parameters in response to experimental modifications of their membrane lipid composition. When determined at 4 degrees C, insulin receptors were present in the control cells at 136,000 sites/cell but this fell to 111,000 (P less than 0.05) in cells enriched in 18:2, and rose to 176,000 (P less than 0.001) in hydroxysterol-grown cells. According to a two-site model, the main effect of 18:2 was a significant increase of the number of high-affinity sites with a concomitant decrease of low-affinity sites. The hydroxysterol had the opposite effects on these parameters. The high-affinity insulin binding capacity of the hepatoma cells was affected by lipid supplementation in a similar way, whether it was determined at 4 degrees C or at 37 degrees C. Assuming a negative cooperativity model, 18:2 enhanced the degree of negative cooperativity among the sites, while 25-hydroxycholesterol reduced it. The time-course of insulin-induced receptor down-regulation was accelerated in the cells enriched in polyunsaturated fatty acids, but reduced in cells exposed to 25-hydroxycholesterol. These insulin-binding alterations cannot be directly related to modifications of cellular growth rate, receptor internalization or membrane fluidity per se, and are discussed as being more likely due to membrane lipid composition than to overall cell metabolism modifications.

Encapsulation of insulin for oral administration preserves interaction of the hormone with its receptor in vitro

It has been shown that insulin associated with nanocapsules of isobutylcyanoacrylate retains biological activity after oral administration to diabetic rats from 6 to 21 days. Because part of this action is unexplained, we focused on the interaction of encapsulated insulin with the insulin receptor in vitro. We have shown that encapsulated insulin is able 1) to bind to insulin receptors both in rat liver plasma membranes and after solubilization from Chinese hamster ovary (CHO) cells transfected with the gene of human insulin receptor, 2) to accelerate 125I-labeled insulin dissociation from its receptor, and 3) to ensure transduction of a signal leading to stimulation of the β-subunit phosphorylation, with parameters similar to those of native insulin. In addition, encapsulated 125I-insulin was rapidly internalized in transfected CHO cells. Analysis of cell-associated radioactivity showed that encapsulated insulin remained largely intact (> 80%) after 3 h, whereas native insulin was mostly degraded. These data indicate that encapsulated insulin fulfills all the earliest events at the receptor level leading to biological actions and suggests that encapsulation protects insulin against insulin degradation inside the cells.

Reversible intramitochondrial release of protein related to unsaturated fatty acids of membranes

Abstract The influence of temperature on intramitochondrial protein and enzyme release was studied in control and “lipid-deficient” rat liver mitochondria and in synaptosomal and “cell body” mitochondria of rat brain. (i) The fatty acid composition of the phospolipid fraction was shown to be different in control and lipid-deficient preparations. (ii) Arrhenius curves for temperature-dependent release of protein showed breaks. (iii) When comparing control to lipid-free rat liver mitochondria and cell body to synaptosomal rat brain mitochondria, shifts in the breaks in the Arrhenius plots were observed for release of aspartate aminotransferase, protein and malate dehydrogenase. (iv) Intramitochondrial temperature-dependent, succinate-induced protein release was also studied in rat liver mitochondria which had previously undergone a succinate-induced release and rebinding cycle. These mitochondria showed a temperature-dependent protein release identical to that of untreated mitochondria.

Succinate transport inhibition by valproate in rat renal mitochondria

Sodium valproate is an antiepileptic drug which may have side effects on different organs. Its mechanism of action, as yet unclear, may involve an effect on membranes. One possibility, an effect on mitochondrial membranes via an inhibition of oxidative phosphorylation, has been studied here by observing the transmembranal transport of a respiratory substrate, succinate, in rat kidney mitochondria incubated with valproate, or from rats injected with valproate. Succinate transport was inhibited in both conditions, which suggests that the effect was probably due to a direct effect of valproate rather than to an action of a valproate metabolite. For the valproate-incubated mitochondria, inhibition, described by a bell-shaped curve, started at a valproate concentration of 10(-7) M and was maximum at valproate 10(-5)M. Valproates effect on mitochondrial transmembranal succinate transport can be compared to other evidence for membranal actions of valproate, actions which may clarify certain therapeutic or toxic properties of this drug.

Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action.

To study the role of transmembrane (TM) domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent‐resistant dimers through TM–TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the β subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin‐induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints.—Gardin, A., Auzan, C., Clauser, E., Malherbe, T., Aunis, D., Crémel, G., Hubert, P. Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action. FASEB J. 13, 1347–1357 (1999)

Metformin modulates insulin receptor signaling in normal and cholesterol-treated human hepatoma cells (HepG2).

The effects of the biguanide anti-hyperglycemic agent, metformin (N,N-dimethyl-biguanide), on insulin signaling was studied in a human hepatoma cell line (HepG2). Cells were cultured in the absence (control cells) or in the presence of 100 microM of a cholesterol derivative, hemisuccinate of cholesterol. Cholesterol hemisuccinate-treatment alters cholesterol and lipid content of HepG2 and modulates membrane fluidity. Cholesterol hemisuccinate-treatment induces a decrease in insulin responsiveness and creates an insulin-resistant state in these cells. Exposure to 100 microM of metformin resulted in a significant enhancement of insulin-stimulated lipogenesis in control and cholesterol hemisuccinate-treated cells. In control cells, metformin altered glycogenesis in a biphasic manner. In cholesterol hemisuccinate-treated cells, metformin inhibited basal glycogenesis but restored insulin-stimulated glycogenesis. Hence, to understand the mechanism of metformin action, we analyzed early steps in the insulin signaling pathway, including insulin receptor autophosphorylation, mitogen-activated-protein kinase and phosphatidylinositol 3-kinase activities, in both control and cholesterol hemisuccinate-treated cells. Overall, the results suggest that metformin may interact with the insulin receptor and/or a component involved in the early steps of insulin signal transduction.

Seasonal variation of the composition of membrane lipids in liver mitochondria of the hibernator cricetus cricetus relation to intramitochondrial intermembranal protein movement

Abstract 1. 1. European hamster ( Cricetus cricetus ) raised under constant temperature conditions show a seasonal variation in the profiles of the structural fatty acids of the liver mitochondrial membrane. In these animals there are but small differences between sleeping and aroused animals in winter. 2. 2. The most important variation in lipid composition occurs in the prehibernating phase—at the end of summer and the beginning of autumn. 3. 3. However, there is no apparent coordinated relation between the seasonal variation of the composition of the fatty acids and the membrane “fluidity” as expressed by the break in the Arrhenius curve for protein release in intermembranal space of the liver mitochondria. 4. 4. This break occurred at a higher temperature in active animals in winter (arousedl than in summer. 5. 5. No general correlation could be found between the breaks in the Arrhenius curves and the variations of the different fatty acid species during the seasonal cycle, except for the most polyunsaturated fatty acid (docosahexanoic acid) where an excellent inverse correlation was observed. 6. 6. Our results suggest that the more fluid parts of the lipidic leaflet of the mitochondrial membrane are those more specifically involved in such phenomena as succinate induced intramitochondrial protein movement and that the changes in composition of the mitochondrial lipids are a possible adaptative advantages for the hibernator.

Inhibition of primary breast tumor growth and metastasis using a neuropilin-1 transmembrane domain interfering peptide

The transmembrane domains (TMD) in membrane receptors play a key role in cell signaling. As previously shown by us a peptide targeting the TMD of neuropilin-1 (MTP-NRP1), blocks cell proliferation, cell migration and angiogenesis in vitro, and decreases glioblastoma growth in vivo. We now explored the clinical potential of MTP-NRP1 on breast cancer models and demonstrate that MTP-NRP1 blocks proliferation of several breast cancer lines including the MDA-MB-231, a triple negative human breast cancer cell line. In models with long term in vivo administration of the peptide, MTP-NRP1 not only reduced tumor volume but also decreased number and size of breast cancer metastases. Strikingly, treating mice before tumors developed protected from metastasis establishment/formation. Overall, our results report that targeting the TMD of NRP1 in breast cancer is a potent new strategy to fight against breast cancer and related metastasis.

Increase of the fluidity of the lipid bilayer of the inner mitochondrial membrane by succinate and phenylsuccinate: A study by EPR and fluorescence

Electron paramagnetic resonance and fluorescence experiments have demonstrated that the lipid matrix of inner membrane of mitochondria was more fluid than the control membrane when incubated with succinate or with one of its non permeant and non metabolizable analog, phenylsuccinate, both of which induce a protein movement from the inner membrane towards the intermembrane space and the inner matrix. Besides, the increase of fluidity seemed more pronounced near the bilayer surface. Although the mechanisms involved in the protein movement are yet unknown, these results lead us to think that they are related to a membrane reorganization involving inter alia the lipid matrix.