Biomedical subjects

François Authier

Publications and source records attributed to François Authier.

6 recordsLinked to original sources

PubMed2006

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.

Adenylyl Cyclases↗

PubMed2006

[Tramadol-induced hypoglycemia. 2 cases].

INTRODUCTION: Tramadol has been approved in France since 1994 for the treatment of moderate to severe pain. The most commonly reported adverse effects involve the central nervous system and gastrointestinal tract. To our knowledge, hypoglycemia has not previously been reported. CASE: We report 2 cases of hypoglycemia related to tramadol, one in a nondiabetic 88-year-old woman, and the other in a diabetic 8-year-old girl. Hypoglycemia resolved after tramadol discontinuation. DISCUSSION: The French Pharmacovigilance (Adverse Drug Reports) database includes several cases of hypoglycemia in patients receiving tramadol. Reports from the literature of dextropropoxyphene-induced hypoglycemia and a pharmacological study of tramadol in rats suggest that the micro opioid receptor is the principal target involved in this hypoglycemic mechanism.

Administration, Oral↗

PubMed2005

Endosomal proteolysis of insulin-like growth factor-I at its C-terminal D-domain by cathepsin B.

IGF-I is degraded within the endosomal apparatus as a consequence of receptor-mediated endocytosis. However, the nature of the responsible protease and the position of the cleavage sites in the IGF-I molecule remain undefined. In vitro proteolysis of IGF-I using an endosomal lysate required an acidic pH and was sensitive to CA074, an inhibitor of the cathepsin B enzyme. By nondenaturing immunoprecipitation, the acidic IGF-I-degrading activity was attributed to the luminal species of endosomal cathepsin B with apparent molecular masses of 32- and 28-kDa. The cathepsin B precursor, procathepsin B, was processed in vitro within isolated endosomes at pH 5 or at 7 in the presence of ATP, the substrate of the vacuolar H(+)-ATPase. The rate of IGF-I hydrolysis using an endosomal lysate or pure cathepsin B was found to be optimal at pH 5-6 and moderate at pH 4 and 7. Competition studies revealed that EGF and IGF-I share a common binding site on the cathepsin B enzyme, with native IGF-I displaying the lowest affinity for the protease (IC50 approximately 1.5 microM). Hydrolysates of IGF-I generated at low pH by endosomal IGF-I-degrading activity and analyzed by reverse-phase HPLC and mass spectrometry revealed cleavage sites at Lys68-Ser69, Ala67-Lys68, Pro66-Ala67 and Lys65-Pro66 within the C-terminal D-domain of IGF-I. Treatment of human HepG2 hepatoma cells with the cathepsin B proinhibitor CA074-Me reduced, in vivo, the intracellular degradation of internalized [125I]IGF-I and, in vitro, the degradation of exogenous [125I]IGF-I incubated with the cell-lysates at pH 5. Inhibitors of cathepsin B and pro-cathepsin B processing, which abolish endosomal proteolysis of IGF-I and alter tumor cell growth and IGF-I receptor signalling, merit investigation as antimetastatic drugs.

Catalysis↗

PubMed2005

Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D.

We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC(50) values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gsalpha protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation.

Animals↗

PubMed2004

Use of high affinity insulin analogues to assess the functional relationships between insulin receptor trafficking, mitogenic signaling and mRNA expression in rat liver.

We have investigated the functional relationships between insulin receptor (IR) trafficking, mitogenic signaling and mRNA expression in rat liver and primary hepatocytes. The low-K(d) insulin analogues [His(A8),His(B4), Glu(B10),His(B27)]-human insulin (-HI) (the H2-analogue), [Asp(B10)]HI and [Glu(A13),Glu(B10)]HI, were studied in liver parenchymal cells and compared with wild-type HI and epidermal growth factor (EGF), a mitogenic inducer. The extent and duration of IR endocytosis were markedly increased in response to the H2-analogue and [Asp(B10)]HI compared to wild-type HI, but similar to HI after [Glu(A13),Glu(B10)]HI administration. Importantly, the insulin analogues induced a higher and more prolonged tyrosine phosphorylation of the IR-beta subunit in endosomes compared to authentic HI. A low cell-free endosome-lysosome transfer of the internalized IR was only observed in response to HI and H2-analogue injection. Concomitant with the low endosome-lysosome transfer of the intact IR-beta subunit, 47 and 50 kDa fragments of the IR-beta subunit accumulated in lysosomal fractions. Neither HI nor the insulin analogues promoted the endosomal recruitment and tyrosine phosphorylation of Shc, whereas EGF accessed the Shc signaling pathway. Moreover, EGF induced a fast and prolonged activation of Raf-1 and MAP-kinase pathways whereas HI and insulin analogues displayed a moderate and transient effect. Finally, treatment of primary rat hepatocytes with HI and the protease-resistant H2-analogue did not affect the total level and relative expression of isotype A and B of IR mRNA regardless of time of exposure. These results suggest a lack of relationship between IR trafficking, endosomal tyrosine phosphorylation and mitogenic signaling in rat liver in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2003

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.

Animals↗