Offspring of protein-malnourished rat dams have permanent alterations in hepatic enzyme activities associated with glucose homeostasis. Hormonal control of hepatic glucose output (HGO) was studied in male offspring of dams fed either a 20% (control) or 8% (low protein) protein diet during pregnancy and lactation. Glucagon (210 pM) stimulated HGO significantly more (P < 0.04) in controls (from 0.72 +/- 0.11 to 3.18 +/- 0.30 mumol.min-1.g liver-1) compared with low-protein animals (from 0.53 +/- 0.11 to 2.05 +/- 0.24 mumol.min-1.g liver-1). Insulin (1 nM) decreased (P < 0.001) HGO in controls to 2.39 +/- 0.37 mumol.min-1.g liver-1 after 10 min but increased HGO (to 2.82 +/- 0.40 mumol.min-1.g liver-1; P < 0.04) in low-protein rats. There were fivefold fewer (P = 0.01) glucagon receptors but a threefold increase (P < 0.05) in hepatic insulin receptor number in the low-protein rats, which was reflected by increased in insulin degradation (P < 0.001). The glucose transporter GLUT-2 was also raised threefold in the low-protein group (P < 0.001). The anomalous response to insulin indicates changes in its metabolic signaling, but normal insulin binding suggests that this alteration is a postreceptor event.

Colonies of cells with distinctive dendritic appearance were observed in methylcellulose cultures of human bone marrow and peripheral blood mononuclear cells (PBMC). Such cells appeared alone in colonies of less than 50 cells, together with macrophages in mixed colonies and also within clusters of T lymphocytes at high culture cell numbers. The morphologic resemblance to lymphoid dendritic cells was confirmed by electron microscopy and the cells were distinguished from macrophages by immunoenzymatic and immunogold labeling with monoclonal antibodies (MoAbs). Like macrophages they were HLA-DR+ and CD4+. However, they lacked nonspecific esterase and the macrophage cytoplasmic marker Y1/82A. Most strikingly, cells were strongly HLA-DQ+ and expressed CD1a (T6), which is characteristic of skin Langerhans cells. Their functional similarity to lymphoid dendritic cells was demonstrated by their ability to stimulate allogeneic mixed leukocyte reactions. Dendritic cell colony numbers were estimated in both bone marrow and peripheral blood of controls and in leukemia and lymphoma patients before and after chemotherapy. Colony numbers were low in control blood and in patients before treatment (less than 1.0 to 3.7/10(5) cells). However, during hematopoietic recovery the mean value increased to 37.5/10(5) cells and this increase correlated closely with the observed increase in circulating colony forming unit-granulocyte macrophage (CFU- GM) in individual patients. Autoradiographic studies demonstrated mitotic activity within CD1a+ colonies and a linear relationship between cultured cells and both pure and mixed colonies was consistent with their derivation from a single precursor. These data indicate that a novel hematopoietic progenitor of dendritic/Langerhans cells (DL-CFU) may now be identified in a clonal assay system and suggest a probable common progenitor for these cells and macrophages.

Insulin signal transduction, initiated by binding of insulin to its receptor at the plasma membrane, activates the intrinsic receptor tyrosine kinase and leads to internalization of the activated ligand-receptor complex into endosomes. This study addresses the role played by the activated insulin receptor within hepatic endosomes and provides evidence for its central role in insulinstimulated events in vivo. Rats were treated with chloroquine, an acidotrophic agent that has been shown previously to inhibit endosomal insulin degradation, and then with insulin. Livers were removed and fractionated by density gradient centrifugation to obtain endosomal and plasma membrane preparations. Chloroquine treatment increased the amount of receptorbound insulin in endosomes at 2 min after insulin injection by 93% as determined by exclusion from G-50 columns and by 90% as determined by polyethylene glycol precipitation (p < 0.02). Chloroquine treatment also increased the insulin receptor content of endosomes after insulin injection (integrated over 0 -45 min) by 31% when compared with controls (p < 0.05). Similarly, chloroquine increased both insulin receptor phosphotyrosine content and its exogenous tyrosine kinase activity after insulin injection (64%; p < 0.01 and 96% and p < 0.001, respectively). In vivo chloroquine treatment was without any observable effect on insulin binding to plasma membrane insulin receptors, nor did it augment insulin-stimulated receptor autophosphorylation or kinase activity in the plasma membrane. Concomitant with its effects on endosomal insulin receptors, chloroquine treatment augmented insulin-stimulated incorporation of glucose into glycogen in diaphragm (p < 0.001). These observations are consistent with the hypothesis that chloroquine-dependent inhibition of endosomal insulin receptor dissociation and subsequent degradation prolongs the half-life of the active endosomal receptor and potentiates insulin signaling from this compartment.Insulin signal transduction is initiated by binding of insulin to its receptor at the plasma membrane, which in turn leads to the rapid autophosphorylation of multiple tyrosine residues on the intracellular portion of the ␤-subunit and the activation of the receptor tyrosine kinase toward exogenous substrates (1, 2). Following autophosphorylation, the activated ligand-receptor complex is internalized into endosomes in liver (3-6) and low density membranes in adipocytes (7,8) and muscle (9). Endocytosis of activated receptors has the twin effects of concentrating receptors within endosomes and allowing the insulin receptor tyrosine kinase to phosphorylate substrates that are spatio-temporally distinct from the plasma membrane (Ref. 10; reviewed in Ref. 11). Subsequent termination of signal transduction is achieved by endosomal insulin degradation (12-16) following dissociation of insulin from its receptor (14, 17) as the intralumenal environment of the endosome acidifies (18). This loss of the ligand-receptor complex attenuates any further ligand-driven recept...

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The effect of chloroquine on the interaction of insulin with its receptor has been investigated under both equilibrium and non-equilibrium conditions. Chloroquine was found to augment insulin binding in a pH-dependent manner between pH 6.0 and pH 8.5, with the maximum occurring at approximately pH 7.0. Analysis of the equilibrium binding data in terms of independent binding sites gave equivocal results but suggested an increase in the high-affinity component. Analysis using the negative co-operativity binding model of De Meyts, Bianco and Roth [J. Biol. Chem. (1976) 251, 1877-1888] suggested that the affinity at both high and low occupancy was increased equally. The kinetics of association of insulin with the plasma-membrane receptor indicated that, although the net rate of association increased in the presence of chloroquine, this was due to a reduction in the dissociation rate rather than an increase in the association rate. This was confirmed by direct measurement of the rates of dissociation. Dissociation was found to be distinctly biphasic, with fast and slow components. Curve fitting suggested that the decrease in dissociation rate in the presence of chloroquine was not due to a decrease in either of the two dissociation rate constants, but rather to an increase in the amount of insulin dissociating by the slow component. It was also found that the increase in dissociation rate in the presence of excess insulin, ascribed to negative co-operativity, could be accounted for by an increase in the amount of insulin dissociating by the faster pathway, rather than by an increase in the dissociation rate constant. Thus chloroquine appears to have the opposite effect to excess insulin, and evidence was found for the induction of positive co-operativity in the insulin-receptor interaction at high chloroquine concentrations. Evidence was also found for the presence of low-affinity chloroquine binding sites with binding parameters similar to the concentration dependence of the chloroquine-induced augmentation of insulin binding.

The cross-linking model for insulin receptor interactions, in which a single insulin molecule may form a cross-link between an insulin receptor's alpha-subunits, has been expressed as a formal compartmental model and subjected to a systematic analysis, examining a number of predictions that have been made for this model. The kinetic parameters for the model were obtained by matching data from insulin receptor equilibrium binding studies and rates of formation of the insulin receptor complex. This analytical study has allowed a clear description of the kinetics of the ligand receptor complexes involved in such a mechanism. We conclude that the cross-linking model accounts for the anomaly of the 10-fold concentration difference in high- and low-affinity binding sites found when insulin binding is analyzed by conventional means. However, the phenomenon of acceleration of dissociation of labeled ligand by unlabeled ligand cannot be accounted for as an intrinsic part of the model. We suggest that this phenomenon arises from the destabilization of cross-link formation when a second insulin molecule binds.

SUMMARY A novel iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, and desferrioxamine were compared for their ability to remove iron and for their site of action in iron release in rats. Repeated intraperitoneal injections of the chelators in rats with widespread tissue labelling by 59Fe derived from transferrin showed comparable "9Fe mobilisation by each chelator in normal and iron loaded rats. Specific labelling of a chelatable "cold" iron pool in hepatocytes by "9Fe derived from ferritin showed this pool to be equally accessible to parenteral doses of both chelators and also to oral 1 ,2-dimethyl-3-hydroxypyrid-4-one, which is an effective oral iron chelating agent that removes iron from parenchymal cells. This and other a-ketohydroxypyridines need further development as potential therapeutic agents in human iron overload.The treatment of iron overload in transfusion dependent thalassaemia using desferrioxamine is expensive' and hence unobtainable in most parts of the world where the disease is common. Furthermore, the need to administer desferrioxamine by continuous subcutaneous infusion often leads to poor compliance with the treatment.2 Consequently, there is a need for a new, cheap, orally active, non-toxic iron chelator. The a-ketohydroxypyridines3 are a promising group of iron chelators which have been shown to remove iron from transferrin,4and ferritin' in vitro, and from mice' 7 and rabbits,8 when administered orally and parenterally.Our studies compared the iron chelating ability of desferrioxamine and 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in rats, being particularly concerned with determining the effect of repeated parenteral doses of the drugs in normal and iron loaded animals; the main site of action of the drugs; and the relative efficiency of oral and parenteral doses of L1. To follow iron mobilisation by the drugs two different tech-

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