Glycosylation defects alter insulin but not insulin-like growth factor I binding to Chinese hamster ovary cells

Insulin binding to two Chinese hamster ovary cell lines with well-defined defects in their glycosylation pathway has been characterized and compared to insulin-like growth factor I (IGF-I) binding in the same cell lines. Insulin competition curves indicate that B4-2-1 cells, which transfer co-translationally to proteins an endoglycosidase H insensitive, truncated lipid-linked oligosaccharide, bind insulin with higher than normal affinity. Lec 1 cells, which fail to process oligosaccharide side chains to complex types, bind with a reduced affinity. The potencies of chicken and guinea pig insulins are appropriate for an insulin receptor in the control (WTB) and both mutant cell lines, whereas rat IGF-II is 3 times more potent than expected in the Lec 1 cells and human IGF-I is less potent than anticipated. Insulin bound to Lec 1 cells dissociates more quickly upon dilution than does insulin bound to either WTB or B4-2-1 cells. The Lec 1 insulin receptor is insensitive to pH change, whereas the other lines show the usual optimum of 8. 125I-IGF-I binds well to all three cell lines and is equally pH-sensitive in all three. Serum from a patient with circulating autoantibodies to the insulin receptor competes for insulin but not IGF-I binding, whereas alpha IR3, a monoclonal antibody directed toward the human IGF-I receptor inhibits IGF-I but not insulin binding. Cross-linking of either 125I-insulin or 125I-IGF-I reveals a typical alpha-subunit in the WTB and B4-2-1 cells but a band with faster mobility in the Lec 1 cells. Insulin (10(-8) M) stimulates autophosphorylation of a beta-subunit in all three lines, but again the Lec 1 subunit demonstrates an anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data demonstrate the differential effect of glycosylation on two closely related receptor molecules.     

Beziehungen zwischen den Endothelzellen der Lebersinusoide und den von Kupfferschen Sternzellen Elektronenmikroskopische Untersuchung

Summary Zymosan given to rats, normal or splenectomized, stimulates the RES of the liver. Under this stimulation, the endothelial cells of the sinusoids change into Kupffer cells. The endothelial cells become hypertrophic and multiply; they acquire progressively the properties of phagocytosis and of digestion shown by the Kupffer cells. Hence both types of cells — the endothelial and the Kupffer cells — belong to the same cellular lineage. Their various aspects result from different functional states.The hyperplasia of the liver RES has no repercussion on the fine structure of the hepatic cells.

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Résumé L'administration de Zymosan à des rats, splénectomisés ou non, provoque une stimulation du SRE du foie. Sous l'effet de cette stimulation, les cellules endothéliales des sinusoïdes se transforment en cellules de Kupffer. Les cellules endothéliales s'hypertrophient et se multiplient; progressivement, elles acquiérent les propriétés de phagocytose et de digestion des cellules de Kupffer. Les deux types de cellules — endothéliales et cellules de Kupffer — appartiennent donc bien à la même lignée cellulaire. Leurs divers aspects correspondent à des états fonctionnels différents.L'hyperplasie du SRE du foie n'a pas de répercussion sur l'ultrastructure des hépatocytes.

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Mit Unterstützung durch den Schweizerischen Nationalfond für die Förderung der Wissenschaftlichen Forschung.

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Stipendiat der Organisation Mondiale de la Santé.Übersetzung von Frau Antje Forssmann, technische Mitarbeit von Max Baumann, Michel Bernard und Charles Moreillon.     

Nitrification and nitrate uptake: Leaching balance in a declined forest ecosystem in eastern France

Nitrate uptake and leaching were measured during one year in a declined fir forest on the Vosges highlands (eastern France), in order to investigate whether excess nitrification could be responsible for a deleterious acidification of the ecosystem. Nitrate uptake by the vegetation was active mainly from spring to early fall, and then reached about 66 kg N ha^-1. No significant leaching loss occurred during the growth period of the vegetation. Significant nitrate leaching occurred in winter (about 17 kg N ha^-1). During fall and winter the nitrification rate was of the same magnitude as values reported for other ecosystems, and, thus, was not considered to be abnormaly strong. No abnormal temporal discoupling of nitrate production and nitrate uptake occurred in the ecosystem, and forest decline must therefore have some other cause.     

Effect of bombesin upon plasma somatostatin-like immunoreactivity, insulin and glucagon in normal and chemically sympathectomized dogs

The present study was designed to determine the effects of intravenously infused bombesin (10 ng/kg/min) upon basal and postprandial plasma somatostatin-like immunoreactivity (SLI), glucagon, insulin and triglyceride levels in normal (n = 12) and chemically sympathectomized (n = 11) dogs. Basal plasma SLI, glucagon and insulin levels rose significantly during the infusion of bombesin in the normal dogs, and this was not altered by chemical sympathectomy. Bombesin infusion enhanced the postprandial SLI response, while attenuating the postprandial glucagon response by 50% and the insulin response in the early postprandial phase of the meal. Sympathectomy did not significantly alter the basal levels of these polypeptides, but augmented the postprandial plasma SLI response during the first 90 min, and reduced the postprandial glucagon response during the infusion of bombesin. The postprandial insulin response was not affected by sympathectomy. In both normal and chemically sympathectomized dogs the rise in postprandial triglyceride levels was attenuated by bombesin infusion.     

Uvomorulin mediates calcium-dependent aggregation of islet cells, whereas calcium-independent cell adhesion molecules distinguish between islet cell types

Rat islets of Langerhans are organized as a core of B-cells surrounded by non-B-cells. It is believed that cell type segregation during histogenesis is the result of the differential expression of cell adhesion molecules (CAMs). Since we have previously shown that in contrast to non-B-cells, homotypic adhesion of pancreatic B-cells is dependent on the presence of Ca2+, the possibility exists that Ca(2+)-dependent CAMs (cadherins) might be in part responsible for islet topography. We now demonstrate that after selective removal of Ca(2+)-independent CAMs from the surface of islet cells by mild trypsin/Ca2+ digestion (TC-treatment), there is no significant difference in homotypic adhesion between sorted B- and non-B-cells in the presence of calcium, suggesting an identical deployment of cadherins. Flow cytometric analysis reveals high levels of uvomorulin on both B- and non-B-cells, without any difference between the two populations. On a "1 to 100" scale, B-cell aggregation in the presence of Ca2+ was decreased by anti-uvomorulin Fab fragments from 67 +/- 4 to 25 +/- 3 (mean +/- SEM, n = 4, P less than 0.01). This level is not different from the degree of B-cell aggregation seen in the presence of 0.5 mM EDTA (22 +/- 2). Aggregation of non-B-cells was only slightly decreased by anti-uvomorulin Fab fragments (from 69 +/- 3 to 52 +/- 4). However, after TC-treatment, homotypic cell aggregation of both B- and non-B-cells was completely inhibited by anti-uvomorulin Fab fragments. Thus, uvomorulin appears to be the only functional cadherin on islet cells, and cell type aggregation properties diverge only by virtue of higher levels of Ca(2+)-independent CAMs on non-B-cells. Fab fragments with the property of perturbing islet cell aggregation in the absence but not in the presence of calcium also prevented pseudoislet organization in vitro, suggesting that Ca(2+)-independent CAMs play the major role in islet cell type segregation. In conclusion, the results show that uvomorulin is responsible for the Ca(2+)-dependent aggregation of islet cells and suggest that the cellular organization within islets or pseudoislets results from the different level of Ca(2+)-independent CAMs on islet cell types.     

Effect of prostaglandin E2 upon release of pancreatic somatostatin-like immunoreactivity

Infusion of prostaglandin E₂ (1 ug/kg/min) in six normal dogs elicited a greater than two-fold rise in pancreaticoduodenal vein somatostatin-like immunoreactivity. Insulin and glucagon also rose. The results raise the possibility that the function of the canine pancreatic D-cell is under prostaglandinergic influence.     

Protein-Protein Interactions in the β-Oxidation Part of the Phenylacetate Utilization Pathway: CRYSTAL STRUCTURE OF THE PaaF-PaaG HYDRATASE-ISOMERASE COMPLEX*

Microbial anaerobic and so-called hybrid pathways for degradation of aromatic compounds contain β-oxidation-like steps. These reactions convert the product of the opening of the aromatic ring to common metabolites. The hybrid phenylacetate degradation pathway is encoded in Escherichia coli by the paa operon containing genes for 10 enzymes. Previously, we have analyzed protein-protein interactions among the enzymes catalyzing the initial oxidation steps in the paa pathway (Grishin, A. M., Ajamian, E., Tao, L., Zhang, L., Menard, R., and Cygler, M. (2011) J. Biol. Chem. 286, 10735–10743). Here we report characterization of interactions between the remaining enzymes of this pathway and show another stable complex, PaaFG, an enoyl-CoA hydratase and enoyl-Coa isomerase, both belonging to the crotonase superfamily. These steps are biochemically similar to the well studied fatty acid β-oxidation, which can be catalyzed by individual monofunctional enzymes, multifunctional enzymes comprising several domains, or enzymatic complexes such as the bacterial fatty acid β-oxidation complex. We have determined the structure of the PaaFG complex and determined that although individually PaaF and PaaG are similar to enzymes from the fatty acid β-oxidation pathway, the structure of the complex is dissimilar from bacterial fatty acid β-oxidation complexes. The PaaFG complex has a four-layered structure composed of homotrimeric discs of PaaF and PaaG. The active sites of PaaF and PaaG are adapted to accept the intermediary components of the Paa pathway, different from those of the fatty acid β-oxidation. The association of PaaF and PaaG into a stable complex might serve to speed up the steps of the pathway following the conversion of phenylacetyl-CoA to a toxic and unstable epoxide-CoA by PaaABCE monooxygenase.