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D B Donner

Publications and source records attributed to D B Donner.

At least 73 records · Page 4Linked to original sources
PubMed1982

Receptor- and non-receptor-mediated uptake and degradation of insulin by hepatocytes.

Native insulin inhibits the binding and degradation of (125)I-labelled insulin in parallel. Half-maximal inhibition of degradation occurs with 10nm-insulin, a hormone concentration sufficient to saturate the insulin receptor. The proportion of bound hormone that is degraded increases as the insulin concentration is increased, suggesting that low-affinity uptake is functionally related to degradation. Since only a small fraction (approx. 10%) of the overall degradation occurs at the plasma membrane, or in the extracellular medium, translocation of bound hormone into the cell is the predominant mechanism mediating the degradation of insulin. In the presence of 0.6nm-insulin, a concentration at which most cell-associated hormone is receptor-bound, chloroquine increases the amount of (125)I-labelled insulin retained by hepatocytes. However, chloroquine increases the retention of degradation products of insulin in incubations containing sufficient hormone (6nm) to saturate the receptor and permit occupancy of low-affinity sites. Glucagon does not compete for the interaction of (125)I-labelled insulin (1nm) with the insulin receptor. In contrast, 20mum-glucagon inhibits 75% of the uptake of insulin (0.1mum) by low-affinity sites. A fraction of the cell-bound radioactivity is not intact insulin throughout a 90min association reaction at 37 degrees C. During dissociation, fragments of (125)I-labelled insulin are released to the medium more rapidly than is intact hormone. The production and transient retention of degradation products of the hormone complicates the characterization of the insulin receptor by equilibrium or kinetic methods of assay. It is proposed that insulin degradation occurs by receptor- and non-receptor-mediated pathways. The latter may be related to the action of glutathione-insulin transhydrogenase, with which both insulin and glucagon interact.

Animals↗
PubMed1982

Consequences of 125I-labeled insulin degradation by hepatocytes on the interpretation of receptor binding studies.

The association of 125I-labeled insulin with hepatocytes was assayed by filtration or microcentrifugation. Assay by centrifugation resulted in a greater amount of retained radioactive label throughout the course of association of 125I-labeled insulin with hepatocytes. Similarly, saturation experiments assayed by microcentrifugation suggested greater binding than filtration. During dissociation, cells isolated by centrifugation release a greater amount of rapid-dissociating radioactive label. Control experiments of [3H]-inulin exclusion with cell pellets, which were isolated during microcentrifugation, demonstrated that the difference between the methods was not due to extracellular trapping of radioactivity. Therefore, the data suggested that there was more low-affinity retention when binding was assayed by centrifugation than filtration. The integrity of the 125I-labeled insulin extracted from hepatocytes was determined by column chromatography. A substantially greater proportion of the extracted radioactivity was fragments of 125I-labeled insulin in cells isolated by centrifugation. It is suggested that the extensive washing of the cells during filtration removes more fragments than does centrifugation. During dissociation, the low-affinity component of radioactivity, which was observed in the centrifugal assay, resulted from the transient retention of insulin fragments. The extensive degradation of insulin, which was assayed by either method, and the differences observed between these methods, should be considered in the interpretation of binding experiments with cells.

Animals↗
PubMed1982

Insulin binding to liver plasma membranes from rats rendered diabetic by alloxan. A kinetic demonstration of two classes of binding sites in equilibrium with each other.

The association of 125I-labelled insulin with liver plasma membranes from diabetic rats was consistent with more than one compartment of binding. After a short association period, insulin dissociation comprised rapid and slow phases. After a long association period, dissociation was only at a slow rate. Lower-affinity hormone-receptor complexes were converted to higher-affinity complexes as the time of occupancy lengthened.

Animals↗
PubMed1982

Affinity change of the adipocyte receptor fails to alter insulin-stimulated glucose transport.

Occupancy increased the affinity of the insulin receptor of the adipocyte. During the affinity change the half-maximal sensitivity of glucose transport to insulin stimulation was unaltered. Decreased maximum response of transport only occurred after the affinity change. There was not a simple relationship between receptor affinity and insulin stimulation of glucose transport in the adipocyte.

Adipose Tissue↗
PubMed1982

Insulin receptors convert to a higher affinity state subsequent to hormone binding. A two-state model for the insulin receptor.

Kinetic experiments were performed to determine the effects of insulin receptor occupancy on insulin binding. The following results were obtained: (a) the rate constant (k1) for uptake of 125I-insulin by liver plasma membranes was 2 X 10(6) M-1 s-1 and invariant at applied hormone concentrations of 7.5 to 100 X 10(-11) M. 125I-Insulin dissociated from membranes in a biphasic manner with rapid (k-1 = 2-4 X 10(-3) s-1) and slow (k-1 = 2-3 X 10(-4) s-1) components of release when dissociation was initiated by dilution into excess medium. Under all dissociation conditions employed, 125I-insulin was the radioactive species bound to and released from membranes. (b) Native insulin (100 nM) or 131I-insulin (5 nM) in the dissociation medium enhanced the dilution-induced dissociation of bound 125I-insulin. In the latter experiment, total receptor occupancy (bound 125I-insulin and 131I-insulin) decreased during dissociation. The enhanced dissociation effect was therefore not necessarily due to increased site occupancy. (c) As association time prior to dissociation was increased, the dissociability of bound 125I-insulin diminished. Decreased dissociability resulted from an increase in the slow component of hormone release at the expense of the rapid component. 131I-insulin was bound to membranes to which 125I-insulin had been prebound. The dissociation of 131I-insulin had been prebound. The dissociation of 131I-insulin was unaffected by the presence of 125I-insulin dissociating at either rapid or slow rates. The data suggest that there are no cooperative interactions between binding sites and that the hormone-receptor complex converts to a higher affinity state subsequent to occupancy since KD = k-1/k1 and K-1 decreased. A two-state model for the hepatic insulin receptor is proposed.

Animals↗
PubMed1981

Up-regulation of insulin receptors in rat liver plasma membranes.

Kinetic experiments (uptake versus time) were utilized to examine the effects of occupancy on insulin receptor availability in rat liver plasma membranes in vitro. The following observations were made: 1) at 4 degrees C, a 3-h exposure of membranes to 100 nM native insulin, followed by removal of unbound hormone, resulted in a subsequent decrease of 125I-insulin binding at 4 degrees C. In a similar experiment at 23 degrees C, no decrease of 125I-insulin binding was observed. 2) At 23 degrees C, 131I-insulin (5 nM) was bound to membranes in a slowly reversible manner after a 3-h association. After removal of free hormone, the 131I-insulin-treated membranes displayed similar binding of 125I-insulin (1 nM) relative to controls despite persistent high level occupancy of receptors by 131I-insulin. 3) At 23 degrees C, phospholipase pretreatment of membranes enhanced 125I-insulin uptake (approximately 40%). Phospholipase-digested membranes exposed to 100 nM native insulin for 3 h bound more 125I-insulin (approximately 40%) than did nondigested membranes preincubated without native insulin. The results allowed speculation that rat liver membranes up-regulated insulin receptors after treatment with insulin and that this was mediated by exposure of cryptic binding sites.

Animals↗
PubMed1980

Formation of a receptor state from which insulin dissociates slowly in hepatic cells and plasma membranes.

125I-insulin dissociated from rat hepatocytes and liver plasma membranes with a time course suggestive of more than a single kinetic process. Dissociation curves were resolved into rapidly and slowly dissociating components. Increasing times of hormone-cell or hormone-membrane incubation prior to the initiation of dissociation increased the proportion of slowly dissociable 125I-insulin and decreased the proportion of rapidly dissociating hormone. The rates of loss of rapidly and slowly dissociating 125I-insulin, 1 to 2 x 10(-3) and 2 to 7 x 10(-5) s-1, respectively, were the same in cell and membrane incubates. The capacity of liver membranes and hepatocytes to bind 125I-insulin in a slowly dissociable state was saturable with respect to insulin concentration (approximately 10(-8) M). The observation of the same physical process in both cells and plasma membranes demonstrates a distinct role for receptors at the exterior surface of target cells in the retention of insulin.

Animals↗
PubMed1980

Characterization of the slowly dissociable human growth hormone binding component of isolated rat hepatocytes.

Human growth hormone (hGH) bound to specific sites on rat hepatocytes. The time course of hGH dissociation was comprised of more than one component. Dissociation was resolved into rapid (t1/2 = 10.5 min) and slow (t 1/2 = 6.4 h) fractions. The amount of slowly dissociable hormone increased for the first 75 min during which time cells and [125I]hGH associated. Subsequently, the amount of slowly dissociable hGH was constant. The time courses of hGH receptor binding and subsequent retention of slowly dissociable label were similar. The capacity of hepatocytes to accumulate slowly dissociable label was saturated by hGH over the same concentration range as the high-affinity binding site (KD approximately 2 nM). This suggested that a receptor-mediated process was responsible for the accumulation of slowly dissociable hGH. Rapidly dissociable label was intact [125I]hGH and fragments resulting from growth hormone degradation. Slowly dissociable hGH recovered from hepatocytes by acid extraction was intact and immunocompetent. There was a large increase in the extent of [125I]hGH degradation between 23 and 37 degrees C. Over this temperature range, the proportion of hGH not in rapid equilibrium with the medium decreased. High concentrations of hGH decreased the amount of slowly dissociable [125I]hGH retained by hepatocytes by competing for high-affinity sites. The interaction of [125I]hGH with low-affinity degradative systems was favored by the presence of hGH. The temperature and concentration dependencies of hGH retention and degradation distinguished these proceses.

Animals↗
PubMed1980

Interconversion between different states of affinity of the human growth hormone receptor on rat hepatocytes: effects of fractional site occupancy on receptor availability.

Isolated rat hepatocytes accumulate a slowly dissociable human growth hormone (hGH) binding fraction with incubation time. Slowly dissociable [125I]hGH is receptor bound, intact and immunocompetent. Fifty-six percent of the bound hormone was slowly dissociable within 3 min of the initiation of hGH-hepatocyte incubation. Subsequently, the proportion of slowly dissociable [125I]hGH increased at the expense of the rapidly dissociable fraction. This suggested that binding induced interconversion between different states of affinity of the hGH receptor. Preincubation with hGH diminished the capacity of hepatocytes to subsequently bind [125I]hGH. Receptor occupancy resulting from accumulation of slowly dissociable hGH accounted for 37 and 62% of the decreased binding after preincubation with 0.79 and 7.9 nM hGH, respectively. Fractional receptor occupancy, among but distinguishable from other processes, may account for the inverse relationship between site number and applied hormone concentration. Addition of hGH to the medium of [125I]-hGH-hepatocyte incubates increased the extent of loss of label from hepatocytes. The progressive retention of intact [125I]-hGH by hepatocytes with site occupancy and invariant receptor affinity subsequent to fractional saturation was inconsistent with negative cooperativity. A mechanism in which hGH diminished reassociation of [125I]hGH with available sites during dissociation was consistent with the available binding data. The interrelationship between peptide hormone in rapid and slow equilibrium with the medium is of fundamental importance in modulating receptor binding and availability.

Animals↗
PubMed1980

REgulation of insulin binding to isolated hepatocytes: correction for bound hormone fragments linearizes Scatchard plots.

Fragments of 125I-labeled insulin (125I-insulin) are rapidly produced after the initial cell binding process. After association of 125I-insulin with hepatocytes, hormone fragments remain bound to cells. At 23 degrees C, approximately 20% of the label bound at steady state was soluble in trichloroacetic acid. Correction of saturation experiments for the presence of bound trichloroacetic acid-soluble insulin fragments decreased the number and increased the affinity of 125I-insulin-binding sites. Label extracted from cell pellets recovered from saturation experiments was characterized by gel filtration; 59%, 55%, 40%, and 36% of the bound label was from intact hormone after recovery from incubation mixtures containing 0.18, 0.60, 4.6, and 7.5 nM applied 125I-insulin, respectively. At high applied 125I-insulin concentrations, the hormone predominantly interacted with lower affinity degradation systems. When binding data were corrected to assay for undegraded 125I-insulin only, curvilinear Scatchard plots were linearized. The insulin receptor is therefore not composed of heterogeneous or negatively cooperative sites. It is necessary to correct for retained fragments of 125I-insulin in order to define mechanisms through which hormone binding and cellular response may be regulated.

Animals↗
PubMed1978

The effects of bioregulators upon amino acid transport and protein synthesis in isolated rat hepatocytes.

Isolated rat hepatocytes prepared by an enzyme perfusion technique possess a functional amino acid transport system and retain the capacity to synthesize protein. Amino acid transport was studied using the non-metabolizable amino acid analog alpha-aminoisobutyric acid. The transport process was time, temperature and concentration dependent. Similarly, leucine incorporation into protein was time and temperature dependent being optimal at 3m degrees C. Amino acid, fetal calf serum, growth hormone and glucose all produced small, reproducible increases in protein synthesis rates. Bovine serum albumin diminished the uptake of alpha-aminoisobutyric acid and leucine incorporation into protein. The amino acid content on either side of the cell membrane was found to affect transport into or out of the cellular compartment (transconcentration effects). High cell concentrations decreased transport and protein synthesis as a result of isotopic dilution of labelled amino acids with those released by the hepatocytes. This was consistent with the capacity of naturally occurring amino aicds to compete with alpha-aminoisobutyric acid for uptake into the hepatocyte. In order to define more precisely the effects of bioregulators on transport and protein synthesis it will be necessary to define and subfractionate cellular compartments and proteins which are the specific targets of cellular regulation.

Amino Acids↗
PubMed1978

Accumulation of a slowly dissociable peptide hormone binding component by isolated target cells.

The overall rate of dissociation and the fraction of bound radioiodinated human growth hormone that dissociated from hepatocytes varied with time of association. A smaller fraction of bound hormone was dissociable from isolated target cells with increased receptor occupancy and increased incubation time prior to the onset of dissociation. The inability of bound label to reequilibrate completely with the medium was demonstrated further by preincubating cells with labeled hormone prior to the initiation of saturation experiments. In such experiments, time-dependent changes in the binding properties of bound label were observed in Scatchard plots, as a result of the inability of prebound label to reequilibrate rapidly with the medium over the time course of such experiments. These data suggest that bound hormone may be distributed between at least two kinetic components. This phenomenon could be interpreted in terms of heterogeneity of sites, a slow conformational change in the receptor, or a model incorporating spatial compartmentalization of sites.

Cells, Cultured↗
PubMed1977

Allosteric interactions between the membrane-bound acetylcholine receptor and chemical mediators: equilibrium measurements.

An approach to equilibrium dialysis measurements has been developed which enables one to study the interaction of chemical mediators with the membrane-bound acetylcholine receptor and to gain information of a type previously obtainable only with soluble proteins. Equilibrium dialysis experiments conducted at pH 7.0,4 degrees C, and mu = 0.18 M, with electroplax membrane preparations from Electrophorus electricus revealed apparently homogeneous binding isotherms for decamethonium with dissociation constants in the range of 0.2-0.4 muM. The following new information has been obtained. (1) The activators of neural transmission, decamethonium and carbamylcholine, occupy overlapping binding sites. (2) These activators and the inhibitors, alpha-bungarotoxin and d-tubocurarine, compete for only one-half of the sites available to them even through the stoichiometry of these is 1:1 as measured with decamethonium (a reversibly binding activator) and alpha-bungarotoxin (an irreversible specific inhibitor). Different receptor molecules, preexisting nonequivalent binding sites, or an allosteric mechanism involving ligand-induced conformational changes are often considered to account for such observations.

Acetylcholine↗