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We have compared the ability of glucagon and three highly purified derivatives of the hormone to activate hepatic adenylate cyclase (an expression of biological activity of the hormone) and to compete with [125]glucagon for binding to sites specific for glucagon in hepatic plasma membranes. Relative to that of glucagon, biological activity and affinity of [des-Asn-28,Thr-29](homoserine lactone-27)-glucagon, prepared by CNBr treatment of glucagon, were reduced equally by 40- to 50-fold. By contrast, des-His-1-glucagon, prepared by an insoluble Edman reagent and highly purified (less than 0.5% contamination with native glucagon), displayed a 15-fold decrease in affinity but a 50-fold decrease in biological activity relative to that of the native hormone. At maximal stimulating concentrations, des-His-1-glucagon yielded 70% of the activity given by saturating concentrations of glucagon. Thus, des-His-1-glucagon can be classified as a partial weak agonist. Highly purified monoiodoglucagon and native glucagon displayed identical biological activity and affinity for the binding sites. Our findings suggest that the hydrophilic residues at the terminus of the carboxy region of glucagon are involved in the process of recognition at the glucagon receptor but do not participate in the sequence of events leading to activation of adenylate cyclase. The amino-terminal histidyl residue in glucagon plays an important but not obligatory role in the expression of hormone action and contributes to a significant extent in the recognition process.
The C-terminal region-sepcific anti-glucagon sera were raised in rabbits using as immunogen, and conjugate of BSA and a C-terminal fragment of pancreatic glucagon. The hapten was prepared by trypsin digestion of the glucagon, which was proved to be a 1:3 mixture of glucagon (18--29) and (19--29). Six rabbits were immunized by subcutaneous injection of an emulsion of the conjugate with complete Freund's adjuvant and five of the rabbits produced antibodies to the glucagon (GC-1, GC-2, GC-3, GC-5 and GC-6). For comparison, rabbit antisera were also produced against glucagon polymer (GA-10) and syrupy glucagon fibrils (PGA-2). All these antisera as well as the pancreatic glucagon-specific antiserum 30 K were characterized with dog gut-extract (gut-GLI) and glucagon-related peptide fragments in the radioimmunoassay systems. The assay systems utilized 125 I-monosubstituted pancreatic glucagon as tracer and human mono-component glucagon as standard. All sera of the GC-series crossreacted with the dog gut-extract very weakly and antisera GC-5 and GC-6 exhibited the lowest crossreactivities with the extract, which were shown to be as low as that of 30k. Characterization of the antiserum GC-5 with purified glucagon related fragments indicated that the major antigenic determinant located exactly in the C-terminal region of glucagon. The present results clearly showed high efficiency of the use of the glucagon C-terminal fragment as hepatenic immunogen in obtaining the C-terminal region-specific, i.e., pancreatic glucagon-specific antisera.
Ten healthy male volunteers were studied to compare the effectiveness of intravenous and subcutaneous injections of 1 mg of glucagon on HG secretion. Plasma HGH level rose to a peak of 6 ng/ml or greater 120 minutes after the subcutaneous injection of glucagon (sc glucagon) in all subjects, whereas the intravenous injection of glucagon (iv glucagon) caused comparable increments in plasma HGH in only six out of ten subjects. Furthermore, in comparison to those in sc glucagon the periods required to show maximum responses were less consistent in iv glucagon. Plasma IRG levels reached a peak of 102.4+/-22.6 ng/ml at two minutes following iv glucagon, and a peak of 3.33+/-1.08 ng/ml at 15 minutes following sc glucagon. These fell to initial levels at 60 minutes and at 180 minutes, respectively. There was no definite correlation either between the magnitudes of changes in plasma IRG and HGH levels or between the velocities of decrement in blood sugar and HGH responsiveness. Judging from its simplicity and reproducibility it may be concluded that sc glucagon is more suitable for a clinical provocative test of HGH release than is iv glucagon. In regards to the mechanism of glucagon-induced HGH release, neither glucagon per se nor the fall of blood sugar after hyperglycemia was assumed to play any major role. The sustained elevation of plasma IRG for a certain period might be responsible for the glucagon-induced HGH release.
The method of producing experimental glucagon deficiency by administration of glucagon antiserum was evaluated in rats. A pool of antisera was prepared, the affinity of which exceeded that of the glucagon receptors of liver cell membranes, whereas the binding capacity of the volume used amounted to more than one-third of the total glucagon content in the rat pancreas. That rapid, extensive, and lasting neutralization of glucagon had taken place after antiserum treatment was indicated by the following findings: When examined more than 1 h after the injection and after 60 min of exercise-stimulated glucagon production, all rats had excess free antibodies in plasma. The concentration of free glucagon was lowered to one-third of the concentration in control rats; at 37 degrees C plasma samples could bind 25% of additional 300 pmol/liter of glucagon in 10 s, and 69% in 120 s; the glycemic response to exogenous glucagon was abolished. Antiserum treatment, however, had no effect on blood glucose in rats fasted for 3 and 10 h, in chemically sympathectomized and adrenomedullectomized rats, and in 48-h-fasted, acutely adrenalectomized rats. The antiserum was found to contain 460 nmol/liter of antibody-bound glucagon, originating in the rabbit in which the antiserum was raised. However, antibody preparations from which the bound glucagon had been effectively removed were equally ineffective in lowering the basal blood glucose in rats, although in three-fourths of the rats the concentration of free glucagon was lowered beyond detection limit. The data indicate that the absolute concentration of glucagon in plasma is of minor importance for the maintenance of basal blood glucose in the rat.
The effects of endogenous and exogenous hyperglucagonemia on the specific binding of glucagon to hepatocyte receptors was studied, as was the response of cAMP to glucagon. In streptozotocin diabetic rats, blood glucose and plasma glucagon increased and plasma insulin decreased as compared with controls. Insulin treatment in diabetic rats restored blood glucose and plasma glucagon toward normal and elevated plasma insulin. Specific binding of (125)I-glucagon to isolated hepatocytes (10(6) cells) decreased in diabetic rats (8.17+/-0.38%) compared to controls (14.05+/-0.87%) and was restored by insulin treatment (12.25+/-0.93%). Specific binding of (125)I-insulin in controls was 7.30+/-10.16%; it increased in diabetic rats to 12.50+/-0.86%, and decreased in diabetic rats after insulin treatment (9.08+/-0.87%). Scatchard analysis and the competition plots of the data indicate that decreased glucagon binding and increased insulin binding in diabetes were due to change in the number of receptors rather than a change in their affinity. Hepatocyte cAMP response to glucagon (0.25-5.0 ng/ml) was almost abolished in diabetic rats and was restored with insulin treatment. Specific glucagon binding by hepatocytes from chronically hyperglucagonemic (glucagon injected) rats was decreased (P < 0.005) to 8.76+/-0.61% compared with controls (13.20+/-0.74%) and acutely hyperglucagonemic animals (13.53+/-1.33%). The decreased binding was associated with a 70% decrease in hepatocyte cAMP response to glucagon compared with a normal response in acutely hyperglucagonemic rats.These data appear to support the concept of receptor regulation by ambient hormone level. In both endogenous and exogenous hyperglucagonemia, however, there was a disproportionately large decrease in cAMP response to glucagon compared to the decrease in glucagon binding.
The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.
UNLABELLED: The mean (+/- SE) peak level of serum growth hormone (GH) after intramuscular injection of glucagon in ten normal adult men was 15.1 +/- 2.1 ng/ml; glucose infusion suppressed the mean peak GH to 9.6 +/- 3.7 ug/ml (p less than 0.05). Pretreatment of eight of these subjects with propranolol caused a modest increase in the mean peak GH after glucagon (19.4 +/- 2.8 ng/ml) but did not improve the mean peak GH after glucagon when glucose was infused (8.7 +/- 2.8 ng/ml). Individual analysis of the peak GH showed that glucose infusion did not uniformly suppress the peak GH after glucagon; in seven subjects the peak GH was suppressed but in three it was not. CONCLUSIONS: (1) The GH response after glucagon is usually due to a fall in serum glucose after the initial rise in serum glucose induced by glucagon. (2) Nevertheless, since glucose does not consistently inhibit the GH response after glucagon, a second mechanism probably exists by which glucagon stimulates GH secretion. (3) Glucose completely suppresses the propranolol-induced increase in the GH response to glucagon; an adrenergic mechanism may be involved in the control of GH secretion by glucose.
Because in the dog, the gastric fundus contains the largest amount of glucagon immunoreactivity (IRG), the IRG of mucosal scrapes of 105 canine stomachs was extracted by acid-ethanol and then precipitated by ether-ethanol. The IRG recovered was measured by antisera 30K, specific for glucagon and K-4023, which cross-reacts with glucagon-like immunoreactivity. Extracts of mucosa of stomach fundus were further purified by gel filtration on Bio-Gel P-30 in 3M acetic acid. One pooled fraction corresponding to marker pancreatic glucagon in its elution volume was then gel-filtered on Bio-Gel P-30 in 0.05 M NH(4)HCO(3) and yielded one IRG peak, which, however, showed three immunoreactive components on polyacrylamide disc gel electrophoresis in urea. In addition, antiserum K-4023 reacted more strongly with that peak than antiserum 30K indicating the presence of glucagon-like immunoreactivity in this fraction. Subsequent ion-exchange column chromatography on DEAE-Sephadex A-25 and then CM-Bio-Gel A allowed purification to a single protein band on disc gel electrophoresis reacting equally to both antisera 30K and K-4023. 1.5 mug of purified gastric glucagon was obtained and its biological effects were compared to those of pancreatic glucagon in isolated rat hepatocytes. When immuno-equivalent amounts (300-2,500 pg/ml) of either type of glucagon were used, the same biological responses with respect to glycogenolysis and gluconeogenesis as well as urea, lactate, and pyruvate production were observed. Liver cyclic AMP was also raised to the same extent by either one of these hormones. We conclude that this moiety of gastric IRG is apparently identical to pancreatic glucagon because (a) their molecular weights, elution properties in ion exchange chromatography, and their electrophoretic mobility are indistinguishable and (b) both hormones elicited identical biological effects in isolated rat hepatocytes.
To determine whether somatostatin inhibits glucagon secretion directly at the pancreatic level and to study quantitatively the relative effects of somatostatin on glucagon and insulin secretion, the effects of various concentrations of somatostatin on glucagon and insulin release from the in vitro perfused rat pancreas in response to arginine (14.2 mM), isoproterenol (2 mg/ml) and theophylline (10 MM) were studied. Glucagon and insulin responses to arginine were progressively inhibited by somatostatin over a concentration range from 0.1-100 ng/ml. At all doses, somatostatin caused greater inhibition of glucagon secretion than of insulin secretion. Approximately 4 ng/ml somatostatin reduced glucagon responses 50%, whereas 90 ng/ml was required to produce comparable inhibition of insulin responses. Glucagon responses to isoproterenol, an activator of adenylate cyclase, and to theophylline, a phosphodiesterase inhibitor, were completely abolished by 100 ng/ml somatostatin. Isoproterenol did cause insulin release in this system, but insulin responses to theophylline were diminished by somatostatin. The present studies thus indicate that somatostatin is a potent inhibitor of both glucagon and insulin secretion and indicate that it acts directly on the pancreatic alpha and beta cells. Glucagon secretion is approximately 20 times more sensitive to the inhibitory effects of somatostatin than is insulin secretion. Furthermore, the present results suggest that somatostatin may act by modifying cAMP-dependent systems rather than by altering cAMP levels.
Immunoreactive glucagon (IRG) fractions from plasma of 8 normal subjects and 4 patients with glucagon secreting tumors were studied by gel filtration techniques on Bio Gel P--30 and Sephadex G--50 columns. The pancreatic glucagon specific anti serum (30K) of Unger was utilized to measure IRG. Columns were calibrated with labelled albumin, proinsulin, insulin and glucagon. Four peaks were defined in normal and tumor bearing patients: peak I (greater than 20 000 mol. wt.), peak II (primarily 9000 mol. wt.), peak III pancreatic glucagon (3500 mol. wt.) and peak IV small gucagon (less than 3500 mol. wt.). Glucagonoma patients differed from our normal and reported normal subjects in that peak II contained most of the circulating IRG. The percent of IRG associated with peak II was 9.5--31.5% in normals and 39.1--61.2% in glucagonomas. Glucagon-like biological activity in an isolated hepatocyte system was demonstrated for all peaks. However, relative to immunoreactivity, peak II showed reduced activity (25--33%). Immunoassay of dilutions of all peaks revealed the probability of immuno determinants identical with procine pancreatic glucagon. The presence of heterogenous IRG peaks with biological glucagon-like activity suggest that the larger molecules may be prohormones. Further, it is possible that specific elevation of peak II may be a diagnostic feature of glucagonomas.
Bilateral kidney exclusion in the anesthetized dog resulted in an immediate and important increase in arterial plasma glucagon. Forty minutes after ligation of the renal arteries, plasma glucagon averaged 200% of the basal values and 90 min after ligation, mean plasma glucagon averaged 357% of the mean basal value. Comparable changes were observed when basal plasma glucagon was markedly suppressed by intravenous infusion of glucose. The rate of production of glucagon by the pancreas was not significantly increased by kidney exclusion. Since the uptake of glucagon by the kidney was previously shown to be quantitatively important, the present findings suggest that abrupt cessation of kidney glucagon uptake is the major factor responsible for the rise in peripheral plasma glucagon levels observed after ligation of renal arteries.
In order to determine whether glucagon levels of diabetic subjects are suppressible, alpha cell responsiveness to acute insulin administration (0.1 units/kg intravenously) was determined in fourteen juvenile onset, healthy diabetic and eight control subjects. In the diabetics, insulin produced a significant but slow fall in blood glucose over 60 min (P less than 0.01). On the other hand, glucagon levels fell dramatically in all diabetics to undetectable levels (P less than 0.001). Only one diabetic became hypoglycaemic and he alone showed a rebound rise of glucagon at 60 min. The rate of fall of blood glucose in the diabetic subjects was not influenced by the basal glucagon level (r=0.13) or the rate of fall of plasma glucagon (r=0.04). The glucose and glucagon responses of control subjects to insulin administration were in sharp contrast to the diabetics: blood glucose levels fell rapidly to hypoglycaemic levels and were associated with a major rise in glucagon levels (mean rise 116 pmol/1, P less than 0.001). We conclude that alpha cell hyperfunction in human diabetes can be completely suppressed by insulin administration and is therefore not autonomous, and that the slow rate of fall of blood glucose following insulin administration in diabetics is not secondary to glucagon excess.
Following a 30 min preincubation in medium containing no isotopes, anglerfish islet tissue was incubated in the presence of [3H]tryptophan and [14C]isoleucine for 20 min. A portion of the tissue was removed for immediate extraction. The remainder was washed thoroughly with unlabeled medium and post-incubated in medium containing an excess of unlabeled tryptophan and isoleucine for varying periods of time. The distribution of radioactive proteins in alcoholic tissue extracts was analyzed by gel filtration and polyacrylamide gel electrophoresis. The distribution of immunoreactive glucagon was determined by radioimmunoassay. Following the 20 min pulse incubation, only proinsulin was labeled with [14C]isoleucine. Two glucagon immunoreactive molecules, one larger than proinsulin (mol wt near 11,400) and the other slightly smaller than proinsulin (mol wt near 9,000), were the primary proteins labeled with [3H]tryptophan following the 20 min. pulse. During chase incubations of increasing duration, 3H-radioactivity appeared in a glucagon immunoreactive molecule with the approximate molecular size of glucagon and increased with chase time while radioactivity in the 11,400 mol wt tryptophan-labeled molecule decreased. With increasing chase time, the 3H-radioactivity attributable to the 9,000 mol wt tryptophan-labeled molecule initially increased and subsequently decreased which is consistent with the pattern that would be expected for a conversion intermediate. The presence of glucagon immunoreactivity in [3H]tryptophan-labeled molecules having molecular weights near that of proinsulin was established by radioimmunoassay of alternate gel slices following electrophoresis of labeled proteins recovered from the proinsulin containing portions of gel filtration eluates. That [14C]isoleucine became incorporated into insulin and [3H]tryptophan became incorporated into glucagon was established by determination of the distribution of radioactivity in polyacrylamide gels following electrophoresis of labeled proteins recovered from the insulin and glucagon containing portions of gel filtration eluates. These results provide preliminary evidence for sequential metabolic cleavage of proglucagon in glucagon biosynthesis.
Although plasma glucagon levels in the rat fetus are in the adult range, hepatic glycogen is present in far greater abundance in the fetus than in the adult. To explain this paradox, adenylate cyclase response to glucagon was studied in partially purified membranes of rat livers obtained throughout perinatal life and at 3 months of age. The adenylate cyclase response to glucagon (10(-9) M) was only 7% of the adult response at day 15 of fetal life and 20% on the 21st day. No until after the 30th day postpartum did not reach maturity. Yet, the adenylate cyclase response to stimulation by NaF was comparable to the adult response throughout fetal life. The binding of [125I]iodoglucagon (2 X 10(-9) M) by these membrane preparations was only 1% of the adult level at day 15 of fetal life and increased to 23% at the 21st day, and, like the adenylate cyclase response to glucagon, did not reach maturity until after the 30th day of postnatal life. In contrast, insulin binding on the 15th day of gestation was 11% of the adult level and on the 21st day 45% of the adult level, reaching adult levels by the 30th postnatal day. An increase in membrane-associated particles, reflecting intramembranous protein, was observed during prenatal life, but the mean particle number per mum2 reached adult levels on the 21st day of fetal life, indicating that subsequent changes in hormone binding were clearly independent of non-specific changes in the number of particles. The findings suggest that the fetal liver is less sensitive to glucagon action than the adult liver, and that this glucagon "resistance" is mediated by a reduced capacity of the hepatocyte to bind glucagon at a time when substantial binding of insulin is demonstrable. Selective discrimination against glucagon may be important in promoting the anabolic processes required for normal fetal development.
The possible existence of circulating pancreatic type glucagon (immunoreactive glucagon as measured with a highly specific antibody) of extrapancreatic origin was investigated in 20-25 kg pigs after pancreatectomy. In intact conscious animals intravenous arginine infusions stimulated glucagon as well as insulin secretion, while blood glucose remained unaffected. Two weeks after pancreatectomy, and 48 hours after insulin withdrawal, basal glucagon and glucose concentrations were elevated (from 22 +/- 3.7 to 55 +/- 9.5 pmol/1 and 5.8 +/- 0.4 to 16.2 +/- 2.0 mmol/l, respectively), (mean +/- SEM), while insulin concentrations were either undetectable or very low. After pancreatectomy, however, glucagon concentrations no longer increased during arginine infusion. Gut type glucagon levels were not affected by pancreatectomy, and did not change during arginine infusion. When examined by gel filtration, plasma from unoperated pigs contained two components of pancreatic type glucagon, one coeluting with the glucagon marker, the other eluting earlier, probably reflecting larger molecular size. After pancreatectomy only this larger component was found in the plasma. The role of this component in the control of blood glucose is unknown.
Nalpha-Trinitrophenyl glucagon was prepared by reaction with trinitrobenzene sulfonic acid and purified by ion-exchange chromatography. This derivative has essentially no ability to activate adenylate cyclase from rat liver nor to increase the levels of cyclic AMP in isolated hepatocytes nor to stimulate protein kinase activity. This derivative also can act as a glucagon antagonist with regard to cyclic AMP production and can decrease the degree of stimulation of adenylate cyclase caused by glucagon, as well as lowering the glucagon-stimulated elevation of cyclic AMP levels in intact hepatocytes. Nevertheless, this derivative is capable of activating glycogenolysis in isolated hepatocytes and in augmenting the effect of glucagon on glycogenolysis. This metabolic effect of the glucagon derivative thus appears to occur independent of changes in cyclic AMP levels. These results suggest that glucagon can also activate glycogenolysis by a cyclic AM-independent process.
To investigate further the "hyperglucagonaemia" of diabetes mellitus, the metabolic clearance rate (MCR) and acute disappearance time (t1/2) of unlabelled pancreatic glucagon were estimated in 9 normal subjects and 7 insulin-dependent diabetics, using a constant infusion technique. The mean MCR (+/-SE) was similar for both groups (control: 9.0 +/- 0.6; diabetics: 11.4 +/- 1.0 ml/kg/min). The MCR was not influenced by the concentration of glucagon at the time of plateau, and the exogenous hormone appeared to be handled similarly to endogenous glucagon. On the other hand, t1/2 calculated from the fractional decay rate of glucagon from plateau was significantly prolonged in the diabetics (t1/2 6.6 +/- 0.5 min) compared with the control group (t1/2 4.8 +/- 0.2 min, P less than 0.01). Furthermore, there was no correlation between MCR and t1/2, for the control, diabetic, or combined group. It therefore appears that the MCR of glucagon is similar in normal and diabetic subjects. However, since the acute disappearance time (t1/2) of glucagon is not identical in these two groups, it appears that the kinetics of the overall in vivo metabolism of pancreatic glucagon are not similar in diabetic and control subjects.