Receptor-mediated degradation of insulin in isolated rat adipocytes. Formation of a degradation product slightly smaller than insulin.

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [TyrA14-125I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [125I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the 'insulin' peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.

The reversible receptor binding of insulin in isolated rat adipocytes measured at 37 degrees C. The binding is not rate limiting for cellular uptake.

The bimolecular binding reaction between mono[TyrA14-125I]iodoinsulin and the insulin receptor was investigated at 37 degrees C in intact isolated rat adipocytes in which membrane traffic was inhibited by 1 mM KCN. This treatment decreased the fraction of cell-associated radioactivity resistant to treatment at pH 3 (usually regarded as internalized ligand) from 70% to 17%. The total amount of tracer being cell-associated at steady state was reduced to about half of the control value partly because of a decreased apparent binding affinity. The t1/2 for the forward reaction was reduced from 414 s in the control cell to 26 s in the KCN treated cell. Likewise, the t1/2 for the dissociation was reduced from 461 s to 67 s. Both rate constants were pH sensitive, the association rate constant being 7-8-fold more than the dissociation rate constant. Since both rate constants for the bimolecular reaction were one order of magnitude greater than those for the uptake and the release of label in the untreated cell, other processes than binding constitute the rate-limiting step(s) in the cellular reaction with insulin.

Uptake and degradation of insulin and alpha 2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways.

The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.

Internalization of insulin and its receptor in the isolated rat adipose cell. Time-course and insulin concentration dependency.

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37 degrees C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [125I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [125I]insulin to these same membrane fractions. At 37 degrees C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20-30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16 degrees C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.

Degradation of the four monoiodoinsulin isomers by the insulin protease of rat liver.

The cleavage of insulin by the partially purified insulin protease was studied using the four [125I]tyrosine-monoiodoinsulins (tyrosine A-14 and A-19 of the A-chain; tyrosine B-16 and B-26 of the B-chain). The rates of conversion of the four isomers to trichloroacetic acid-soluble form was in the order B-26 greater than A-14 greater than A-19 greater than B-16. The following was observed in experiments which gave 19/14/5/3 percent conversion to trichloroacetic acid-soluble products: the loss of ability to bind to IM-9 lymphocytes was approx. 55% for all four isomers. About 70% of the radioactivity was in the 'insulin' peak, and about 30% was in peptides smaller than insulin as judged by gel filtration on Sephadex G-50. The descending limb of the 'insulin' peak contained significant amounts of radioactive material not binding to IM-9 lymphocytes. This material showed multiple peaks when applied to high performance liquid chromatography. Other experiments were designed to cause an almost complete degradation of the isomers. Under these conditions, the radioactivity eluted on Sephadex G-50 largely as iodotyrosine (and some small peptides) using the A-14, B-16 and B-26 isomers, whereas iodotyrosine was absent using the A-19 isomer. Thus, the insulin protease appears to first degrade insulin to multiple products with molecular sizes slightly smaller than insulin and subsequently to small peptides (e.g containing tyrosine A-19) and amino acids (e.g. tyrosine A-14, B-16 and B-26).

Evidence for binding of human pregnancy zone protein-proteinase complex to alpha 2-macroglobulin receptors.

125I-labelled human pregnancy zone protein complexed with chymotrypsin was removed from the circulation with a half-time of 2.3 min after intravenous injection in rats. After 6 min about 67% of the label was present in the liver and about 3% was in the spleen, both in male and in female pregnant rats. The half-time of removal was more than 30 min for native pregnancy zone protein. Uptake into other organs, including placentae and feti, was negligible. 30 pM labelled pregnancy zone protein X chymotrypsin was specifically bound to rat hepatocytes and adipocytes and to human fibroblasts and monocyte-derived macrophages at 4 degrees C. Binding was almost completely abolished by a saturating concentration of unlabelled alpha 2-macroglobulin X trypsin. Binding of 15 pM labelled macroglobulin complex was completely abolished by a saturating concentration of pregnancy zone protein X chymotrypsin. In rat hepatocytes, binding of pregnancy zone protein complex was lower than that of alpha 2-macroglobulin complex at low ligand concentrations. Half-maximal receptor occupancy was obtained with about 300 pM pregnancy zone protein complex. Unlabelled alpha 2-macroglobulin or pregnancy zone protein complex failed to accelerate dissociation of the labelled pregnancy zone protein complex under conditions where dissociation of alpha 2-macroglobulin was markedly enhanced. It is concluded that pregnancy zone protein and alpha 2-macroglobulin complexes bind to the same receptors. The quantitative differences may be related to the fact that alpha 2-macroglobulin is a tetramer whereas the functional unit of pregnancy zone protein is probably a dimer.

Hagfish insulin: the discrepancy between binding affinity and biologic activity.

Hagfish insulin exhibits a binding affinity of about 25% of that of pig insulin in rat adipocytes and IM-9 lymphocytes, even though its relative biologic potency is only about 5% in adipocytes. The dissociation rate constant of hagfish insulin is about half that of pig insulin, and the association rate constant is about one eighth. A longer time is, therefore, required for hagfish insulin to reach a steady state of binding. Failure to reach steady state is the probable reason why some previous results suggested a relative binding affinity of hagfish insulin of the same magnitude as the relative biologic potency.

[125I]diiodoinsulins. Binding affinities, biologic potencies, and properties of their decay products.

UNLABELLED: Insulin was iodinated with 0.3-0.4 mol 125I/mol insulin using the lactoperoxidase method. About one-third of the radioactivity incorporated into insulin was in diiodoinsulins and about 40% of these molecules contained diiodotyrosine in residue 14 of the A chain. Most of the remaining molecules contained one A14-monoiodotyrosine and one monoiodotyrosine in either position A19, B16, or B26. The binding affinity and biologic potency of this heterogeneous diiodoinsulin preparation was not significantly different from that of A14-[125I]monoiodoinsulin in rat adipocytes, whereas it was slightly reduced in hepatocytes and IM-9 lymphocytes. From the iodine distribution and previous data on the binding affinity of each of the four monoiodoinsulin isomers it was calculated that A14-diiodotyrosine-insulin possesses full binding affinity and biologic potency in adipocytes. Diiodoinsulins isolated from another iodoinsulin preparation (iodate method) contained 58% A19-diiodotyrosine-insulin, and most remaining molecules contained one A19-monoiodotyrosine. The binding affinity of this mixed diiodoinsulin preparation was approximately one-fourth of that of A14-monoiodoinsulin in adipocytes, IM-9 lymphocytes, and hepatocytes. It was calculated that A19-diiodotyrosine-insulin is nearly devoid of binding affinity. The diiodoinsulins (lactoperoxidase method) decayed to iodide (probably from diiodotyrosine-insulin) or to polymers with little specific but a markedly increased nonspecific binding. In addition, the polymers had a marked tendency to adsorb to cellulose acetate filters. CONCLUSIONS: 1. The binding affinities of diiodoinsulins range from very low values to values at least as high as that of insulin depending on the positions of the iodine moieties. 2. The relative binding affinities vary among tissues. 3. Polymeric decay products give high nonspecific binding.

Tyrosine A14[125I]monoiodoinsulin: Preparation, Biologic Properties, and long-term stability.

125I-insulin was prepared by reacting 17.4 nmol porcine insulin (100 micrograms) with 5 mCi 125I (about 2.4 nmol) using the lactoperoxidase method. The reaction product was subjected to gel electrophoresis and the band containing A14 [125I]monoiodoinsulin was eluted. This preparation showed a specific activity of about 1.5 Ci/mumol as evaluated by radioimmunoassay and bioassay, i.e., about 75% of the theoretical maximum. The content of radioactive derivatives other than A14 monoiodoinsulin was less than 2%. The binding affinity of tracer A14 monoiodoinsulin to adipocytes, hepatocytes, and cultured human lymphocytes was twice as high as that of A19 monoiodoinsulin. Binding to antibodies was examined to 10 guinea pig anti-insulin sera. Three sera did not distinguish between the two tracers, whereas seven exhibited higher binding of the A14 tracer. A detailed analysis of one of the discriminating sera showed that the average affinity constant was about 2.5 times lower for the A19 tracer than for the A14 tracer. The A14 monoiodoinsulin tracer is remarkably stable. After 200 days the specific activity had declined to about half of its original value which is consistent with the hypothesis that the physical decay of [125I]monoiodoinsulin (T 1/2 equals 60 days) extinguishes the activity of the molecule without causing major damage of other molecules. By this time 96% of the radioactivity migrated with insulin when subjected to gel filtration on Sephadex G-50, 4% was in the void volume, and nothing in the total column volume or later. Binding to receptors was indistinguishable from that obtained at time zero. It is concluded that Tyr A14[125I]monoiodoinsulin represents an advance in biologic work as compared with previous tracers for insulin.

The biologic potency and binding affinity of biosynthetic human insulin in isolated rat adipocytes.

Using the isolated rat adipocyte system, we could not detect any difference in binding affinity or biologic potency of biosynthetic and pancreatic human insulin.

Uptake of rat and human alpha 2-macroglobulin-trypsin complexes into rat and human cells.

Uptake of rat and human alpha 2-macroglobulin-trypsin complexes was measured in rat hepatocytes, rat and human adipocytes and human fibroblasts. Uptake and degradation of 125I-labelled rat complex were about one-third of that of the human complex in the various isolated cell types. In rat hepatocytes, the apparent Km for cell association of the rat complex was about 16 nM as compared to about 6 nM for the human complex. The Vmax values were similar, about 1 X 10(4) molecules X cell-1 X min-1. Thus, rat alpha 2-macroglobulin (an acute-phase protein) complexed with trypsin follows the same pathways of uptake as the human homologue, although with a somewhat lower affinity for the uptake system.

Isolation of rat peritoneal mononuclear and polymorphonuclear leucocytes on discontinuous gradients of Nycodenz.

Centrifugation of rat leucocytes from thioglycollate-induced inflammatory peritoneal exudate on a discontinuous gradient of Nycodenz with a density of 1106 g/l and an osmolarity of 400 mosmol/l separated the polymorphonuclear from the mononuclear leucocytes. The cells on the interphase between the buffer and the gradient medium contained 96% mononuclear leucocytes with a recovery of greater than 60%, and the bottom fraction consisted of 98% polymorphonuclear leucocytes with a yield of 91%, when an exudate isolated 20 h after the injection of thioglycollate was fractionated. Leucocytes isolated from non-inflamed rat peritoneum could be enriched in the fraction of nonspecific esterase-positive cells from 86% to 96% with a recovery of 82% on a gradient with a density of 1091 g/l and an osmolarity of 325 mosmol/l. The viability of the isolated cells was greater than 95% (trypan blue exclusion test), and there was no measurable reduction in the fraction of phagocytosing cells (latex and opsonized zymosan) after exposure to the hypertonic gradient material.

Characterization of the binding of bovine thrombin to isolated rat hepatocytes.

Isolated rat hepatocytes possess per cell 4,500 high-affinity binding sites for thrombin with a Kd of 30-40 pM, and 2.8 X 10(5) low-affinity sites with a Kd of 30 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labelled thrombin is achieved after 3 min at 37 degrees C and 7 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 1-2 X 10(-2) s-1 and 3-4 X 10(-4) s-1. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radioactivity migrates as intact thrombin upon sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3-treatment. Cell-associated radioactivity dissociated from the cells binds just as well in a receptor assay as tracer incubated in a conditioned medium under the same conditions, indicating the absence of a quantitatively important receptor-mediated degradation of the ligand.

Receptor-mediated degradation and internalization of insulin in the adenocarcinoma cell line HT-29 from human colon.

In the adenocarcinoma cell line HT-29 receptor-bound insulin is substrate for a proteolytic process leading to the release of about half of the cell-associated [125I]monoiodoinsulin in the form of [125I]iodide and [125I]monoiodotyrosine. Classical lysosomal inhibitors (NH+4, methylamine, leupeptin) did not inhibit this proteolysis. Inhibitors of membrane traffic (chloroquine and monensin) and of metabolism (CN-) inhibited the fractional receptor-mediated degradation. The former led to an increased cell-associated 125I activity whereas the latter reduced the uptake. Sulphydryl reagents inhibited the receptor-mediated degradation. The data are not compatible with a quantitatively major role of lysosomes in the receptor-mediated insulin degradation. However, since the process requires energy it is suggested that the receptor-mediated degradation takes place in vesicles other than secondary lysosomes. The responsible enzyme(s) may belong to the thiol group of proteases. Both insulin and the insulin receptor are internalized as a consequence of incubation of HT-29 cells with insulin.

Receptor-mediated degradation of human growth hormone in rat adipocytes and cultured human lymphocytes (IM-9).

In cultured human lymphocytes (IM-9) and in isolated rat adipocytes human growth hormone is substrate for a receptor-mediated degradation. When the cells are incubated with monoiodinated human growth hormone half of the radioactivity dissociating from the cells is in the form of [125I]monoiodotyrosine. Since IM-9 lymphocytes have no receptor-mediated degradation of insulin, obviously insulin and human growth hormone follow different pathways in this cell type. In the rat adipocyte colchicine and monodansylcadaverine caused quantitatively different uptake and degradation of these 2 ligands suggesting that also in this cell type the pathways are functionally different. The application of different inhibitors suggests that the receptor-mediated degradation of growth hormone in these 2 cell types takes place in an acidified compartment by an energy-requiring process and involving thiol groups.

The mechanism of receptor-mediated degradation of insulin in isolated rat adipocytes: indirect evidence for a non-lysosomal pathway.

Receptor-bound insulin is substrate for a degradation leading to the release of about half the cell-associated [125I]monoiodoinsulin as [125I]monoiodotyrosine. Classical lysosomal inhibitors of the amine type (cloroquine, methylamine and NH+4) only partly inhibited this receptor-mediated degradation. Leupeptin, which is very effective in other systems, was without any effect in the present system. The degradation could not be reduced by lowering the ATP content of the cells. Sulphydryl reagents strongly inhibited the degradation as has also been shown for the cytosolic insulin-specific protease. Microtubules and microfilaments are probably not involved since inhibitors of the cytoskeleton were without marked effects. It is suggested that in the rat adipocyte only a minor part of the receptor-mediated degradation of insulin takes place via the classical endocytotic lysosomal pathway.

The cellular dynamics of hepatic receptors for alpha 2-macroglobulin.protease complex and for insulin are different.

Making freshly isolated rat hepatocytes permeable by 0.4 g/liter digitonin doubled the number of binding sites for alpha 2-macroglobulin.trypsin complex without changing the affinity. Thus, digitonin unmasked a receptor pool, probably of intracellular origin. The total cellular binding capacity was measured in the presence of digitonin, the surface-exposed in its absence. Upon preincubation of the cells at 37 degrees C, the total cellular binding capacity for alpha 2-macroglobulin.trypsin decreased over a 2-h period to 0.26 of the initial value. By contrast, the surface-exposed binding capacity initially increased in response to a preincubation at 37 degrees C, reached after 20 min a peak value 1.74 times that at 0 time, followed by a decrease. Neither the increase in nor the loss of surface-exposed binding capacity was influenced by inhibitors of lysosomal functions, protein synthesis and glycosylation. Colchicine abolished the increase in surface-exposed binding capacity but not the disappearance. By contrast, phenylarsine oxide (inhibitor of endocytosis), N-ethylmaleimide, and phenylmethanesulphonyl fluoride inhibited the receptor loss, suggesting that the loss occurred by proteolysis. The insulin receptor concentration, studied in parallel, remained practically constant in the investigated period in the presence and absence of digitonin. Thus, the hepatic receptor for alpha 2-macroglobulin.protease complexes is regulated independently of other specialized plasma membrane proteins.

[Early clinical exposure--an instant success. The new medical curriculum at the University of Aarhus]. / Tidlig klinik--en øjeblikkelig succes. Beskrivelse of en ny studieordning på Aarhus Universitet.

INTRODUCTION: In the new medical curriculum at the University of Aarhus, a third term, 20-week course focussing on early patient contact was launched. MATERIAL AND METHODS: Nine prototypical and clinically important disease entities each formed the basis of one-week courses covering an introductory clinical lecture, presentation of "paper" cases, and formalised training of pertinent clinical skills. This was integrated with plenaries and group work in physiology pertaining to the disease and the patient cases. In addition, seminars were held in patient-doctor relationships, and environmental and social medicine. Introductory lectures were given on topics, such as medical ethics, taxonomy of diseases, the organisation of hospital-based health care. At the end of the term, the students resided for eight weeks at county hospitals, which do not traditionally participate in pregraduate teaching. Each student followed one particular patient, which formed the basis of a written essay. RESULTS: Early clinical lectures (87 +/- 8%, mean +/- SD) and use of clinical cases (73 +/- 8%) were well received by third term students, and 87% found that the "paper" cases facilitated their understanding of physiology. The evaluation of the hospital training was very positive (rated excellent or good by > 95%). DISCUSSION: We conclude that early introduction to clinical practice is feasible and well received by the students.