The role of insulin dissociation from its endosomal receptor in insulin degradation.

Mechanisms that terminate signals from activated receptors have potential to influence the magnitude and nature of cellular responses to insulin. The aims of this study were to determine in rat liver endosomes (the subcellular site of insulin signal termination) whether dissociation of insulin from its receptor was a pre-requisite for ligand degradation and whether the state of receptor phosphorylation influenced the dissociation and hence endosomal degradation of insulin and/or receptor recycling. Following in vivo administration of 125I-[A14]-insulin or analogues (native, X10 or H2, relative binding affinities 1:7:67) livers were removed and endosomes prepared. In the endosomal preparations a significantly greater percentage of both analogues were receptor-bound than native insulin with concomitantly less ligand degradation. When rats were injected with protein-tyrosine phosphatase inhibitors (peroxovanadium compounds bpV(phen) or bpV(pic)) before insulin, endosomal insulin receptor phosphotyrosine content, assessed by Western blotting, was increased as was receptor-bound 125I-[A14]-insulin, whilst insulin degradation was decreased. Peroxovanadiums also completely inhibited recycling of insulin receptors from endosomes. However, treatment of freshly isolated endosomes with acid phosphatase which completely dephosphorylated the insulin receptor, did not return the rate of insulin dissociation and degradation to control values, suggesting that peroxovanadium compounds elicit their effect on binding and degradation via a mechanism other than as protein-tyrosine phosphatase inhibitors. We conclude that promotion of sustained receptor binding decreases endosomal insulin degradation and extends the half-life of the activated endosomal receptor, which in turn would be expected to potentiate insulin signalling from this intracellular compartment.

Chloroquine extends the lifetime of the activated insulin receptor complex in endosomes.

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 insulin-stimulated 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 receptor-bound 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.

An evaluation of the cross-linking model for the interaction of insulin with its receptor.

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.

Altered regulation of hepatic glucose output in the male offspring of protein-malnourished rat dams.

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.

Chloroquine augments the binding of insulin to its receptor.

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.

Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow.

Colonies of CD1a+ HLA-DR+/DQ+ CD4+ cells with the functional and some of the structural attributes of Langerhans cells are observed in human bone marrow cultures in semi-solid media and are assumed to be the progeny of an early progenitor, the dendritic/Langerhans cell CFU (CFU-DL). The cytokine-regulated growth of these cells has been studied using a chemically defined serum-free system to culture both unfractionated and highly enriched bone marrow progenitor cell populations. Although unfractionated cell growth was optimal in serum replete cultures with PHA-stimulated leukocyte-conditioned medium (PHA-LCM) suboptimal proliferation of CFU-DL was observed in serum even in the absence of PHA-LCM. No colonies were observed under serum-free conditions when granulocyte-macrophage CSF (GM-CSF), IL-3, granulocyte CSF (G-CSF), and macrophage CSF (M-CSF) were present at levels optimal for granulocyte colony-forming unit (CFU-G) and macrophage colony-forming unit (CFU-M) growth. Addition of IL-1 alpha to these cytokines stimulated a small number of CFU-DL. However, in the presence of GM-CSF and IL-3, TNF-alpha or TNF-beta (5 U/ml) were both highly effective in promoting growth up to 82% of optimal and CFU-G growth was also enhanced at these concentrations. TNF was only active during the first 3 days of culture and higher concentrations of TNF-alpha but not TNF-beta were inhibitory for both CFU-DL and CFU-G. CD34+ cell-enriched populations were also enriched for both myeloid progenitors (CFU-G + CFU-M) and CFU-DL to 36- and 48-fold, respectively, and single cell cultures of CD34+ cells yielded single colonies containing both CD1a+ dendritic cells and CD1a- macrophages. Thus dendritic/Langerhans progenitors in the bone marrow expresses CD34, have a capacity for both macrophage and dendritic cell differentiation, and depend on hemopoietic growth factors and TNF for their further development in vitro.

Identification of hematopoietic progenitors of macrophages and dendritic Langerhans cells (DL-CFU) in human bone marrow and peripheral blood.

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.

Iron chelation studies using desferrioxamine and the potential oral chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, in normal and iron loaded rats.

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 59Fe mobilisation by each chelator in normal and iron loaded rats. Specific labelling of a chelatable "cold" iron pool in hepatocytes by 59Fe 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 alpha-ketohydroxypyridines need further development as potential therapeutic agents in human iron overload.

The effects of ethanol and anticonvulsants on erythroid delta-aminolaevulinic acid synthase.

The activity of delta-aminolaevulinic acid (ALA) synthase was measured in bone marrow from 13 control subjects, 12 chronic alcoholic patients and 9 patients on long term anticonvulsant therapy. The majority of patients in both groups had macrocytic red cells in the absence of megaloblastic changes in the marrow. There was a significant increase in ALA synthase activity in the alcoholic patients but no significant increase in enzyme activity in the patients on anticonvulsants and overall there was no correlation of activity with MCV. The macrocytosis associated with these drugs does not therefore appear to result from accelerated erythroid haem synthesis.

In-vitro studies of ineffective erythropoiesis in rheumatoid arthritis.

Ineffective erythropoiesis was assessed in a series of 32 patients with rheumatoid arthritis by means of a new in-vitro method which measures the release of haem from a labelled cohort of erythroblasts in culture. Haem release was significantly increased in patients with the anaemia of chronic disorders but was normal in those who were not anaemic or who had an iron-deficiency anaemia. In 2 patients with anaemia of chronic disorders haem release returned to normal after successful antirheumatic therapy. The increased ineffective erythropoiesis in patients with rheumatoid arthritis and anaemia of chronic disorders appeared to be unrelated to functional iron deficiency and was not attributable to a serum factor.

A simple in vitro method for the assessment of ineffective erythropoiesis.

A simple in vitro method has been developed for the assessment of ineffective erythropoiesis by measuring the release of heme from a labeled cohort of erythroblasts in short-term suspension culture. The release of labeled heme was shown to correlate with the death of erythroblasts in culture determined by cell counting. Heme release was markedly increased in conditions where there is known to be excessive ineffective erythropoiesis, while in hematologic disorders where ineffective erythropoiesis is thought to be normal, heme release was within the normal range.

Determination of delta-aminolaevulinic acid synthase activity in human bone marrow using high performance liquid chromatography.

A high performance liquid chromatographic (HPLC) method is described for the rapid and specific determination of the activity of the enzyme delta-aminolaevulinic acid synthase (ALA-S) in mitochondria prepared by sonication of human bone marrow cells. After incubation with 14C-alpha-ketoglutarate the 14C-delta-aminolaevulinic acid (ALA) formed is converted to a pyrrole derivative, 2-methyl-3-carbethoxy-4-(3-propionic acid) pyrrole. This is isolated by reversed-phase ion-pair chromatography on a Hypersil-SAS column with methanol-water (45:155, v/v) in the presence of 0.005 mol/l 1-heptanesulphonic acid (PIC B-7) as the mobile phase. The radioactivity of the isolated pyrrole is determined by scintillation counting. The optimal substrate concentration and pH were 0.17 mmol/l alpha-ketoglutarate and pH 7.4, with an optimal period of sonication of 18s. Under these conditions ALA production was proportional to the concentrations of erythroblasts in the initial sample and was linear with time up to 60 min. The addition of pyridoxal phosphate (PLP) did not affect ALA-S activity in normal subjects. The mean ALA-S activity in 10 haematologically normal control subjects was found to be 318.8 pmol.10-6 erythroblasts.h-1 (S.D. 125.8, range 193-444.6).

Folate-dependent serine synthesis in lymphocytes from controls and patients with megaloblastic anaemia: the effect of therapy.

The utilization of [14C]formate for serine synthesis by lymphocytes was impaired in all the patients with pernicious anaemia and in 70% of other patients with megaloblastic anaemia. In pernicious anaemia this was corrected by vitamin B12 therapy in 48 h but not by folate therapy although patients given folate showed a satisfactory haematological response.