Degradation Of Glucagon Research Articles

125I] Glucagon Binding by Liver Membranes from Young and Adult Rats

We have previously shown that adipocytes from adult (between four and five months old) rats have reduced glucagon binding and glucagon-stimulated lipolytic activity when compared with cells from young (1.5 months old) animals. In the present study we measured specific [125I] glucagon binding by purified liver plasma membranes isolated from young and adult rats. When expressed on the basis of membrane protein content, 5'-nucleotidse activity, or specific [125I] insulin binding, the extend of [125I] glucagon binding by liver membranes was not influenced by aging. Furthermore, the degree of [125I] glucagon degradation was the same in both membrane preparations. These data describe a unique condition in which glucagon binding and hormone sensitivity diminish in one tissue but remain unaltered in another.

Development of glucagon sensitivity in neonatal rat liver.

The ontogenesis of the hepatic glucagon-sensitive adenylate cyclase system has been studied in the rat. With a partially purified liver membrane preparation, fetal adenylate cyclase was less responsive to glucagon than the enzyme from neonatal or adult livers. Similar results were obtained in gently prepared liver homogenates, suggesting that destruction of essential components of the fetal liver membrane did not account for the relative unresponsiveness of the adenylate cyclase enzyme to glucagon. Investigation of other factors that might account for diminished fetal hepatic responsiveness to glucagon indicate (a) minimal glucagon degradation by fetal membranes relative to 8-day or adult tissue; and (b) available adenylate cyclase enzyme, as suggested by a 13-fold increase over basal cyclic AMP formation with NaF in fetal liver membranes. These results indicate that neither enhanced glucagon degradation nor adenylate cyclase enzyme deficiency accounts for the relative insensitivity of the fetal hepatic adenylate cyclase system to glucagon. In early neonatal life, hepatic adenylate cyclase responsiveness to glucagon rapidly developed and was maximal 6 days after birth. These changes were closely paralleled by a fivefold increase in glucagon binding and the kinetically determined Vmax for cyclic AMP formation. These observations suggest that (a) fetal hepatic unresponsiveness to glucagon may be explained by a limited number of glucagon receptor sites; (b) during the neonatal period, the development of glucagon binding is expressed primarily as an increase in adenylate cyclase Vmax; (c) the ontogenesis of hepatic responsiveness to glucagon may be important in the resolution of neonatal hypoglycemia.

Insulin and glucagon degradation by the kidney I. Subcellular distribution under different assay conditions

Insulin and glucagon degradation by rat kidney homogenates and subcellular fractions was examined under a variety of conditions including high and low substrate concentrations, at pH 4 and pH 7, with and without glutathione. At high insulin concentration (4.1 · 10 −5 M) insulin degradation by the homogenate was greatest at pH 4 but at low insulin concentration (1 · 10 −10 M) insulin degradation was greatest at pH 7. At either high or low glucagon concentration glucagon degradation by the homogenate was greatest at pH 7. Glutathione at pH 7 stimulated insulin degradation at high insulin concentrations and inhibited insulin degradation at low concentrations. Glucagon degradation at pH 7 was inhibited at both high and low concentrations of glucagon by glutathione. Separation of kidney into cortex and medulla prior to homogenation produced a pattern of insulin and glucagon degradation identical to the whole homogenate but glucagon degradation by the medulla was greater than by the cortex. Examination of degradation by subcellular fractions revealed that at high concentration at neutral pH most insulin was degraded by the 100 000 × g pellet but at low insulin concentrations over 90% of the activity was in the 100 000 × g supernatant. At pH 7, at both high and low concentrations, most glucagon-degrading activity was in the 100 000 × g pellet, although the cytosol also had activity. At pH 4 most degradation occurred in the lysosomal fractions. Separation into cortex and medulla again showed similar distribution of activity as the whole gland with the medulla having more glucagon-degrading activity than the cortex. With low insulin concentrations the cortex 100 000 × g supernatant had higher relative specific activities than the medulla supernatant. Examination of recoveries of enzyme activity revealed that the subcellular fractions consistently had markedly less insulin-degrading activity than the original homogenate. This loss of activity was only discernible when insulin degradation was performed at pH 7 at low substrate concentrations. Comparable losses of glucagon-degrading activity were not seen.

Insulin and glucagon degradation by the kidney II. Characterization of the mechanisms at neutral pH

Examination of insulin and glucagon degradation by rat kidney subcellular fractions revealed that most degrading activity was localized to the 100 000 × g pellet and 100 000 × g supernatant fractions. Further characterization of the degrading activities of the 100 000 × g pellet and supernatant suggested that three types of enzymatic activity were present at neutral pH. From the cytosol an enzyme with characteristics of the insulin glucagon protease of skeletal muscle was purified. This enzyme appeared to be responsible for insulin degradation by the kidney at physiological insulin concentrations. This enzyme also contributed to glucagon degradation but was not the most active mechanism for this. In the 100 000 × g pellet at least two separate enzymatic activities were present. One of these had properties consistent with those described for glutathione insulin transhydrogenase and appeared to be responsible for insulin degradation at high insulin concentration. The other enzyme was associated with the brush border and had properties consistent with the brush border neutral protease. This enzyme appeared responsible for glucagon degradation at both low and high substrate concentrations. An apparent marked synergism between the 100 000 × g pellet and the 100 000 × g supernatant was noted for insulin degradation at physiological insulin concentrations. Pellet glucagon-degrading activity and soluble insulin-degrading activity were necessary for this. The mechanism was found to be limited insulin degradation by the soluble enzyme resulting in both trichloroacetic acid-precipitable trichloroacetic acid-soluble fragments followed by further degradation of the fragments by the glucagon-degrading enzyme resulting in an additional increase in trichloroacetic acid-soluble products.

The Effect of Lithium on Unstimulated and Glucagon‐stimulated Urinary Cyclic AMP Excretion in Rat and Man

Abstract The purpose of this study was to determine whether lithium also inhibits hormone‐stimulated adenylate cyclase in vivo. The effect of long‐term lithium treatment on unstimulated and glucagon‐stimulated cyclic AMP excretion was studied in the rat and in man. The influence of lithium on plasma glucagon degradation was also investigated. It was found that in the rat lithium doubled unstimulated and glucagon‐stimulated urinary cyclic AMP excretion. In lithium treated rats plasma glucagon concentrations thirty minutes after intraperitoneal injection were twice that of the control rats. In man, lithium affected neither cyclic AMP excretion nor glucagon degradation. These results offer no support for the hypothesis that in vivo lithium in general inhibits hormone‐stimulated adenylate cyclase. However, in the intact organism lithium may have additional pharmacological actions, and complex regulatory mechanisms which may modify the cyclic AMP metabolism. Therefore it may be premature to conclude that lithium per se does not have an inhibitory action on glucagon‐sensitive adenylate cyclase in vivo.

Sites of cleavage of glucagon by insulin-glucagon protease

To study the mechanism of degradation of glucagon with purified insulin-glucagon protease, glucagon was reacted with the enzyme at various times of incubation. The proteolysis was followed by the production of flourescamine-reacting material as well as reaction with dansyl chloride, cleavage by acid hydrolysis, and identification by thin layer chromatography. For quantitative measurement of the degradation products, [ 14C] dansyl derivatives were produced, identified by autoradiography, and counted. In the degradation products in addition to histidine, the dansyl derivatives of tyrosine, phenylalanine, two leucines, alanine and lysine were identified. For comparison, glucagon was also reacted with chymotrypsin and the degradation products consisted of threonine, serine, two leucines and valine. Thus, insulin-glucagon protease degrades glucagon in a manner distinct from that of chymotrypsin.

The heterogeneity and degradation of glucagon studied by polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis has been used to study the heterogeneity of glucagon, and changes in composition resulting from thermal degradation. The same technique can be employed, as a limit test, to monitor the amounts of insulin and desamidoglucagon in glucagon intended for therapeutic use.

Proteolytic degradation of insulin and glucagon

The degradation of insulin and glucagon by a highly purified enzyme isolated from rat skeletal muscle was investigated. A sensitive assay for proteolytic degradation of insulin and glucagon using fluorescamine to detect an increase in primary amine groups was established. As measured by an increase in fluorescamine reactive materials, insulin was rapidly degraded by this highly purified enzyme without requiring initial disulfide cleavage. Associated with the increase in fluorescamine reactive materials was a decrease in immunoassayable insulin. Glucagon was also proteolytically degraded by this enzyme but a number of other peptides and proteins including proinsulin, and A and B chains of insulin were not degraded. Thus, we have demonstrated that insulin (and glucagon) can be proteolytically degraded by an enzyme isolated from an insulin sensitive tissue, skeletal muscle. Proteolytic degradation by this enzyme requires the intact insulin molecule rather than separate A and B chains.

Insulin and glucagon degradation by the same enzyme.

An insulin-degrading enzyme has been previously isolated from rat skeletal muscle and highly purified by a series of procedures including affinity chromatography on insulin-agarose. Glucagon-degrading activity was also found in each of the purification steps including the final highly purified material. Insulin- and glucagon-degrading activities were associated with the same band on polyacrylamide gel and both activities were obtained from affinity chromatography on glucagon-agarose. Insulin- and glucagon-degrading activities could not be differentiated by the effect of sulfhydryl reagents, pH, or by heat inactiva-tion. Insulin served as a competitive inhibitor of glucagon degradation (Ki = 1.5 × 10−8M) and glucagon was a competitive inhibitor of insulin degradation (Ki = 5.3 × 10−6M). On the basis of these studies it appears that insulin and glucagon can be degraded by the same enzyme which is found in the soluble fraction of rat skeletal muscle homogenate.

Studies of glucagon resistance in large rat adipocytes: 125I-labeled glucagon binding and lipolytic capacity

This study is concerned with potential modifications of large fat cells from adult rats (400-450 g) that make them resistant to stimulation by glucagon. The lipolytic capacity and (125)I-labeled glucagon-binding capability of these cells were compared with these properties of small glucagon-sensitive cells from young rats (130-160 g). As determined by maximal stimulation with theophylline, dibutyryl cAMP, or epinephrine, the lipolytic capacity of large cells was not markedly different from small cells, which suggests that an alteration contributing to glucagon insensitivity is not present in the enzymes involved with hormone-mediated lipolysis. Glucagon-binding studies did indicate a difference between the two cell types. Both large cells and particulate fractions from large cells bound less (125)I-labeled glucagon than small cells or small-cell particles. That diminished binding is not a consequence of glucagon degradation is indicated by the similar amounts of (125)I-labeled glucagon degraded by both cell types. The decrease in (125)I-labeled glucagon binding was not as marked as the decrease in lipolytic response to glucagon stimulation. This lack of correlation and the relationship between elevated phosphodiesterase levels and glucagon insensitivity described in the accompanying report suggest that diminished binding explains only in part the marked resistance to glucagon found in large cells.

Human placental transfer of glucagon.

1. The permeability of human chorion laeve in vitro to 125I-labelled glucagon was investigated. The problem caused by degradation of 125I-labelled glucagon in the preparation and a means to prevent this are described. 2. The human chorion laeve was found to be impermeable to iodinated glucagon.

A possible role for the adenyl cyclase system in calcitonin release.

SUMMARY A possible role of the adenyl cyclase system in calcitonin (CT) release has been investigated by measurement of the rate of CT secretion in isolated porcine thyroid glands perfused in situ. Dibutyryl cyclic AMP increased the rate of secretion of CT and this effect was enhanced by theophylline. Concentrations of glucagon as low as 1·4 nm (5 ng/ml) induced a significant increase in the rate of CT secretion when precautions were taken to prevent enzymic degradation of glucagon. This stimulatory effect of glucagon on CT secretion was also increased by theophylline. Significant stimulation of CT release was demonstrated with adrenaline in the presence of α-adrenergic blockade with phentolamine and could be inhibited by β-adrenergic blockade with propranolol. In contrast, thyrotrophin, tri-iodothyronine, serotonin, parathyroid hormone and cyclic guanosine monophosphate had no significant acute effects on CT secretion. It is suggested that the C-cells of the thyroid gland contain an adenyl cyclase system similar to that described in other tissues and that physiological concentrations of glucagon and catecholamines may influence the response of the C-cell to plasma calcium concentration.

Inactivation and Degradation of Glucagon by Dipeptidyl Aminopeptidase I (Cathepsin C) of Rat Liver: INCLUDING A COMPARATIVE STUDY OF SECRETIN DEGRADATION

Chloride and sulfhydryl activators in glucagol degradation and secretion by purified rat liver dipeptidyl aminopeptidase I /Cathepsin C/

Control of Gluconeogenesis in Liver: II. EFFECTS OF GLUCAGON, CATECHOLAMINES, AND ADENOSINE 3′,5′-MONOPHOSPHATE ON GLUCONEOGENESIS IN THE PERFUSED RAT LIVER

Glucagon, epinephrine, norepinephrine, and cyclic AMP stimulated gluconeogenesis from lactate in perfused livers from fasted and fed rats. The stimulation was most marked when these agents were added to livers from fasted rats in which gluconeogenesis had become partially inactive during a preliminary perfusion without substrate. It is suggested that inactivation is consequent to the degradation of endogenous glucagon and catecholamines. Inactivation could be prevented by high levels of lactate. On a molar basis, glucagon was about 100-fold more effective in stimulating gluconeogenesis than the catecholamines, and 100,000 times more effective than exogenous cyclic AMP. Gluconeogenesis was as sensitive as glycogenolysis to stimulation by any of the agents and was as rapidly activated. It is proposed that cyclic AMP mediates the action of glucagon and catecholamines on gluconeogenesis. Glucagon stimulated gluconeogenesis from lactate and pyruvate but not from dihydroxyacetone or fructose, suggesting a point of action in the gluconeogenic pathway between pyruvate and triose phosphate. The maximum rate of gluconeogenesis from oxalacetate was about the same as that from pyruvate. Because lactate production from oxalacetate was substantial and the same tissue level of malate was found with pyruvate or oxalacetate, it appeared that oxalacetate was not metabolized as such by the liver but only after breakdown to pyruvate. Ethanol stimulated gluconeogenesis and lactate formation from pyruvate. The effects of ethanol and glucagon were additive indicating that glucagon does not act by increasing NADH supply. The gluconeogenic effect of glucagon was observed with concentrations of lactate in the physiological range. It is proposed that glucagon plays a physiological role in the maintenance of the blood glucose through control of gluconeogenesis as well as glycogenolysis in the liver. Sympathetic nervous activity, leading to the intrahepatic release of catecholamine, may also play an important role in increasing glucose output and lactate uptake by the liver, particularly during exercise or hypoglycemia.

Glucagon clearance by the isolated perfused rat liver.

SummaryIn the isolated perfused rat liver the biological half-life for pork immunoassayable insulin was determined to be 17 min. The corresponding figures for beef-pork, rat pancreatic, and rat gut, immunoassayable glucagon were 19, 23, and 27 min, respectively. The data are in keeping with the liver, which is a major site for degradation of insulin and glucagon.

Effect of glucose concentration on insulin and glucagon release from isolated islets of Langerhans of the rat.

The isolated islets of Langerhans of the rat have been utilized to study simultaneous insulin and glucagon release. There is very little degradation of insulin or glucagon when a proteolytic enzyme inhibitor, Trasylol, is added to the incubation vessels. Glucose exerts an opposite effect on insulin and glucagon release. As the glucose concentration in the incubation media is raised from 30 to 300 mg. per 100 ml., there is a decreased secretion of glucagon and an increased secretion of insulin. The addition of 2-deoxyglucose to the media inhibited insulin release but had no effect on glucagon. The results are discussed in reference to the role of glucagon in insulinogenesis and in regard to the possible mechanisms of insulin and glucagon release.