Search

The role of glucagon in diabetic hyperglycaemia has been a matter of controversy because of difficulties in the production of selective glucagon deficiency. We developed a high-capacity (40 nmol/ml), high-affinity (0.6 x 10(11) l/mol) monoclonal glucagon antibody (Glu-mAb) and gave i.v. injections (4 ml/kg) to rats in order to study the effect of selective glucagon deficiency on blood glucose. Controls received a mAb against trinitrophenyl. Glu-mAb completely abolished the hyperglycaemic effect of 2.86 nmol/kg glucagon in normal rats (p < 0.05, n = 6). In moderately hyperglycaemic rats injected with streptozotocin as neonates (N-STZ), Glu-mAb abolished a postprandial increase in blood glucose (from 11.2 +/- 0.7 mmol/l to 17.3 +/- 1.8 mmol/l in controls vs 10.5 +/- 0.9 mmol/l to 9.3 +/- 1.0 mmol/l; cross-over: n = 6, p < 0.05). No significant effect of Glu-mAb treatment was observed in more hyperglycaemic N-STZ rats (cross-over, n = 4) and in severely hyperglycaemic rats injected with STZ as adults (n = 6), but after insulin treatment of the latter, at doses partially restoring blood glucose levels (12.7 +/- 4.3 mmol/l), Glu-mAb administration almost normalized blood glucose (maximal difference: 6.0 +/- 3.8 mmol/l; cross-over: n = 5, p < 0.05). In conclusion, our results provide strong additional evidence for the hypothesis that glucagon is involved in the pathogenesis of diabetes. The hormone plays an important role in the development of STZ-diabetic hyperglycaemia, but glucagon neutralization only leads to normoglycaemia in the presence of insulin.

A potent high-titre glucagon antibody pool was used to induce a state of acute glucagon deficiency in order to investigate the importance of glucagon in maintaining euglycaemia in the fed and fasted anaesthetised rat. Binding characteristics of the antiserum and evidence of its neutralisation of the biological effects of exogenous glucagon are described. The amount of antibody administered was capable of neutralising up to 12 times the total content of glucagon (approximately 1 nmol) in the rat pancreas. The hyperglycaemic response to 1.43 nmol exogenous glucagon was significantly inhibited in the rat by glucagon antibodies given intravenously or intraperitoneally (p less than 0.001). However, no changes in plasma glucose occurred in rats fasted 16 h (4.35 +/- 0.1 mmol/l or 24 h (4.0 +/- 0.05 mmol/l) after antibody administration. The same dose of glucagon antibodies produced no change in plasma glucose (6.1 +/- 0.2 mmol/l), immunoreactive insulin (1.85 +/- 0.05 microgram/l) or immunoreactive somatostatin (110 +/- 30 ng/l) in rats after antibody administration. Antibody excess, equivalent to a binding capacity for glucagon of 40 nmol/l in the plasma of recipient animals, was demonstrable at all times after passive immunisation. The absence of any affect on glucose concentrations following immunoneutralisation of glucagon suggests that glucagon secretion may not be a major factor in the maintenance of euglycaemia in the rat.

Fasting (24 h) normal dogs and depancreatized dogs were injected intravenously with highly purified porcine insulin (Actrapid) in the doses of 0.2 U/kg and 0.5 U/kg, respectively. Blood glucose decreased from 152 +/- 41 (SEM) mg/100 ml to 39 +/- 7 mg/100 ml in the depancreatized dog and from 95 +/- 3 mg/100 ml to 42 +/- 4 mg/100 ml in the normal animal. Using a specific antiserum for "pancreatic" glucagon, the circulating level of glucagon immunoreactivity did not rise from the basal value of 247 +/- 31 pg/ml in the depancreatized group whereas it rose significantly from 223 +/- 24 pg/ml to 321 +/- 41 pg/ml in the normal group. In contrast intravenous infusion of 7 g of arginine increased "pancreatic" glucagon immunoreactivity in both groups. Thus, extrapancreatic glucagon of the pancreatic type does not respond to hypoglycaemia but to arginine infusion.

Plasma immunoreactive glucagon, C-peptide and substrates (glucose, lactate, and alanine) were measured in 21 pancreatectomized patients and 28 patients with chronic calcifying pancreatitis during arginine infusion. Results were compared with those obtained in control and in insulin-dependent diabetic subjects, and in pancreatectomized subjects receiving a combined infusion of glucagon and arginine or somatostatin and arginine. Plasma immunoreactive glucagon in the pancreatectomized patients was 230 +/- 26 pg/ml (control subjects 100 +/- 13 pg/ml, p less than 0.001), but was unchanged following arginine or somatostatin. Following ethanol extraction of plasma it became undetectable. Similar results were obtained in patients with chronic pancreatitis. In contrast to the insulin-dependent diabetic subjects, no changes in blood glucose, lactate, and alanine concentrations were found during arginine infusion in the pancreatectomized or pancreatitis patients. Addition of glucagon restored the metabolic response to arginine in the pancreatectomized patients. Our results confirm previous smaller studies that in pancreatectomized patients, A cell function is absent or insignificant.

Biogel P-30 filtration of plasma from Type 1 (insulin-dependent) diabetic patients and normal subjects in basal state and after an oral glucose load was assayed with a C-terminal (30 K) and a glucagon-like immunoreactivity-cross-reacting antiserum (R8). Up to four immunoreactive peaks of approximate molecular sizes of greater than 20,000 (fraction I), 9000 (fraction II), 3500 (fraction III) and 2000 (fraction IV) were detected with the two antisera in both groups. In the basal state, the only significant difference observed between both groups was a higher R8-reactivity in fraction II in the group of diabetic patients, although the R8 minus 30 K values for this fraction did not show a significant difference between both groups. After glucose the only significant differences were an increase of R8-reactivity in fraction II in both groups (p less than 0.01) and a decrease of 30 K-reactivity in fraction III (IRG3500) in normal subjects (p less than 0.05). In seven out of 12 diabetic patients, 30 K-reactivity in fraction II (IRG9000) and III (IRG3500) increased above their basal values. The gut-glucagon-like immunoreactivity response to oral glucose (delta R8-delta 30 K values in fraction II) was similar in both the diabetic and normal subjects. These results indicate that the paradoxical rise in plasma immunoreactive glucagon after oral glucose in diabetic patients may be due to an increase of both IRG3500 and/or IRG9000, the gut-glucagon-like immunoreactivity released during glucose absorption has a molecular weight of approximately 9000, and no differences in plasma gut-glucagon-like immunoreactivity were observed in Type 1 diabetic patients when compared with normal subjects, either in the basal state or after glucose ingestion.

The sera of 30 patients who had been treated with conventional beef insulin were tested for binding of insulin and other pancreatic hormones. All showed antibody binding of insulin, 29 binding of proinsulin, 29 binding of pancreatic polypeptide, two binding of glucagon but none of the sera bound vasoactive intestinal peptide or somatostatin. After changing therapy to highly purified pork insulin the binding capacity of sera for insulin and the other hormones was monitored for up to 35 months and a steady fall was found in nearly all cases. In eight of the patients conventional beef insulin treatment was resumed: in one month binding of insulin and of the other hormones increased back to the initial levels. In eighteen subjects who had only received highly purified pork insulin low levels of insulin binding were found with no binding of proinsulin or other hormones. The amounts of proinsulin and contaminating hormones in highly purified pork insulin are so low that they are not immunogenic; conventional beef insulin not only contains immunogenic amounts of proinsulin and the contaminating hormones pancreatic polypeptide and glucagon but also is more immunogenic than purified pork insulin.

Analysis of the plasma from a totally pancreatectomized patient, with antiserum 30 K, has demonstrated basal glucagon immunoreactivity (GIR) levels in the normal range (80-110 pg/ml). Neither i. v. arginine nor oral glucose affected these GIR values, thus indicating the absence of functioning pancreatic or gastrointestinal A-cells. Furthermore, filtration of whole plasma on Bio Gel P-30 showed no GIR in the 3500 MW elution volume. GIR was found to be distributed in two peaks. One peak eluted in the protein region, similarly to "big plasma glucagon" (BPG), and the second peak appeared after the glucagon-I125 marker. The protein-sized moiety was not absorbable by charcoal, and on Sephadex G-100 it eluted within the globulin region. When subjected to trypsin treatment, it yielded smaller GIR fractions. According to these criteria, it can be assumed that this component is identical to BPG. Therefore, an extrapancreatic source for BPG is suggested. On the other hand, the presence of fasting hyperglycaemia in this patient indicates that insulin deficiency by itself suffices to raise blood sugar to diabetic levels.

The effects of glucose and arginine on insulin secretion in the presence of glucagon antibodies were investigated in rats in vivo. In contrast to controls, animals given glucagon antibodies showed an inhibition of arginine-stimulated (p less than 0.001), but not glucose-stimulated, insulin secretion. That these effects were not due to incomplete neutralisation of endogenous glucagon is evidenced by the presence of large antibody excess throughout the duration of the experiments. Both the glucagonotropic effect of arginine (319 +/- 60 ng/l, p less than 0.01) and the insulinotropic effect of exogenous glucagon (8.3 +/- 0.8 microgram/l, p less than 0.001) were demonstrable under our experimental conditions in the absence of exogenous glucagon antibodies. These observations suggest that different mechanisms are involved in the stimulation of insulin release by arginine and by glucose, and that glucagon may play an important physiological role in the mediation and regulation of insulin secretion by secretogogues, such as arginine.

The protein synthesis in normal and in EMC-virus infected mouse islets of Langerhans was investigated. Mouse large glucagon immunoreactivity was determined by an immunoassay after chromatographic separation. It was characterized as a peptide of 16 000 MW with in intracellular half-life of 35-45 min. The proportional reduction of void volume proteins, large glucagon immunoreactivity and proinsulin synthesis after infection shows, that both alpha- and beta-cells are damaged by the virus. A reduction in the synthesis of the three protein fractions was already found 6 hrs after inoculation of the virus and remained nearly constant for 48 hrs. An almost complete breakdown of protein synthesis occurred 60 to 70 hrs after infection and was paralleled by the first light microscopic changes in the islets. The stimulation of proinsulin synthesis by glucose was preserved for 48 hrs after EMC-virus infection.

Uraemia was induced in pigs by ligation of the renal vascular pedicle, and uraemic plasma was analysed for glucagon and glucagon-related peptides. A preponderance of large molecular weight (Mr) components comprising glicentin and moieties of slightly lower Mr was found, accounting for 73 +/- 3% (mean +/- SEM, n = 12) of the total plasma glucagon-like immunoreactivity. Comparisons with glicentin 1-61, produced by controlled, stepwise, consecutive digestion of purified natural glicentin with carboxypeptidases (carboxypeptidase A followed by carboxypeptidase B, and again by carboxypeptidase A and B), gel filtration, ion exchange chromatography, reverse phase HPLC and radioimmunoassays for the glucagon sequences 6-15 and 19-29 and for the glicentin sequence 12-30 all indicate that glicentin 1-61 constitutes approximately 57% of the large Mr glucagon-related peptides found in uraemia in pigs.

Total immunoreactive glucagon (IRG) and immunoreactive glucagon of A cell origin (IRGa) were measured in the serum of normal, sham-operated and depancreatized rats, after the administration of three glucagon antagonists: insulin (5--200 mU/rat/h), somatostatin (SRIF; 100 microgram/kg/h) and antiglucagon serum (AGS, enough to bind three times the calculated total amount of circulating IRG). Since no differences were noted between the responses of normal and sham-operated animals, the values were pooled and used as controls. Pancreatectomy caused a significant increase in serum glucose, IRGa and total IRG and a significant decrease in serum insulin. AGS and SRIF significantly decreased serum glucose in control, but not in depancreatized rats, even though SRIF caused a significant decrease of IRGa in all animals. SRIF significantly decreased plasma insulin in control rats, but did not modify total IRG secretion in either groups. In control rats the minimum effective hypoglycaemic dose of insulin (5 mU/rat/h) may have decreased serum IRGa, but not total IRG. At higher doses (20 mU/rat/h) insulin stimulated glucagon secretion. In depancreatized animals, higher doses of insulin (200 mU/rat/h) were needed to lower serum glucose. On the other hand, a dose of 100 muU/rat/h was sufficient to lower the serum IRG. We conclude that although hyperglucagonaemia may contribute to the hyperglycaemia of the untreated depancreatized rats, the excessive secretion of glucagon is secondary to insulin insufficiency and that, at least in this animal model, the hypoglycaemic action of insulin is only minimally dependent upon its ability to suppress glucagon secretion.

Pancreatic islet cell antibodies and 12 other autoantibodies were measured at the time of diabetes diagnosis in 46 Pima Indians, aged 17--47 years, and in 46 age-sex matched non-diabetic controls. Islet cell antibodies were found in only two diabetics, aged 20 and 25, compared with none of 46 controls. Neither of the subjects with islet cell antibodies had other autoantibodies. At least one type of autoantibody was found in 14 (30%) of the diabetics and in 14 controls, but none was significantly associated with diabetes. This study indicates that diabetes in the Pima Indians, even those with an onset below 25 years of age, is almost entirely of type II, in that the disease is not associated with islet cell antibodies, ketoacidosis, or insulin dependence.

The results reported in the literature regarding glucagonaemia in genetically obese fa/fa rats are conflicting: normal, increased or decreased plasma glucagon levels have been reported. Due to the existence of several molecules endowed with glucagon-like immunoreactivity, it was thought that the conflicting data could be related to the degree of specificity of the different glucagon antibodies. Three antibodies that all qualified as being specific for pancreatic glucagon were used. It was found that, depending on the antibody, absolute values of basal glucagonaemia or arginine-induced glucagon output varied quantitatively and qualitatively in both lean and obese rats. When non-extracted basal or stimulated plasma samples were passed on a G-50 Sephadex column, glucagon-like immunoreactivity was present over a wide range of molecular weights, indicating the presence of non-pancreatic glucagon molecules. When an ethanol extraction was used, the fractions eluting from the G-50 Sephadex column contained only pancreatic glucagon immunoreactivity. It is concluded that ethanol extraction is necessary for the measurement of the 3500 daltons glucagon. Using this methodology it was found that: (1) basal glucagonaemia was low but identical in the two groups of rats; (2) arginine-induced glucagon secretion was greater in obese than in lean animals; (3) glucagonaemia was decreased by glucose administration in lean but not in obese rats. It is concluded that there are, in obese animals, dysfunctions of glucagon output that may play a role in their abnormal glucose tolerance.

Different techniques for the extraction and initial purification of porcine gastrointestinal glucagon-like immunoreactivity (GLI) were compared with reference to yield, and preservation of number and pattern of GLI components. The conventional acid-ethanol technique combined with ethanol-ether purification gave high yields and a reproducible pattern of components. Large amounts of tissue were more easily extracted using another technique, based on extraction by boiling, extraction and precipitation with acetone, and--if necessary--salting out. By means of the latter two techniques mucosal tissue from all of the porcine gastrointestinal tract was extracted and subjected to gel filtration. Glucagon-like peptides were searched for using: 1. a radioimmunoassay which quantifies gut type glucagon (GTG), as well as pancreatic type glucagon (PTG), 2. a radioimmunoassay highly specific for pancreatic type glucagon (PTG), and 3. a radioreceptor assay based on binding of glucagon to porcine liver cell membranes. The oesophageal, the fundic, and the antro-pyloric parts of the gastric mucosa contained very small amounts of GLI. The cardiac gland region contained small amounts of a peptide indistinguishable from "true" glucagon. The duodenal mucosa contained small amounts of "true" glucagon and may be a smaller, glucagon-like peptide. The mucosa of the small intestine contained large amounts of both high and low molecular weight GTG and, in addition, PTG of high molecular weight and "true" glucagon. The colon also contained these components with "true" glucagon in high concentrations. Only small GTG and "true" glucagon were receptor-active, the former with less than its immunometric potency.

Five patients with mild maturity-onset diabetes were given 250 ml of a 20% glucose solution by intraduodenal infusion and eight other patients similarly received an amino acid solution in a dose of 0.5 g amino acids per kg body weight. The pancreatic and gut glucagon-like immunoreactivity (pancreatic GLI and gut GLI) in plasma were measured before and after the application of the two stimuli. Each person was tested twice; the first (control) test was followed by a second test after three days of treatment with phenformin 150 mg daily, plus the same 150 mg dose taken 60 min before the intubation. The plasma pancreatic GLI increased slightly during both infusions, but was not affected by phenformin. Intraduodenal infusion of both glucose and the amino acid solution induced a greater rise in plasma gut GLI. After treatment with phenformin, the fasting plasma gut GLI was higher than the control value in eleven of thirteen patients. In most cases higher gut GLI plasma levels were also found after duodenal administration of glucose and amino acids. These data furnish further evidence of the local action of antidiabetic biguanides on the intestinal wall, including its hormonal activity. The hypothesis is advanced that the phenformin-induced increase in gut GLI secretion may bring about competition of the latter with pancreatic glucagon for receptors in liver cell membranes, reducing the effect of glucagon on the liver, and thus contributing to a decrease in glycaemia.

Proliferation of adipose tissue adjacent to intrasplenic transplants of whole isogeneic neonatal rat pancreas has consistently been noted. In this study over a period of 18 months there was a progressive increase in the amount of fatty tissue in the vicinity of surviving transplants. Immunohistochemistry demonstrated the presence of insulin, glucagon, somatostatin and pancreatic polypeptide within islet cells in long term grafts. Electron microscopy demonstrated a close association between islets and lipid droplets. Ductal elements within the transplants survived and showed close association with endocrine cells, but exocrine pancreatic tissue degenerated rapidly. Radioimmunoassay of extracts from surviving transplants in isogeneic rats confirmed the presence of high levels of insulin and glucagon after transplantation. In contrast, allogeneic intrasplenic transplants of rat pancreas failed to survive and showed no evidence of adipose tissue proliferation. Furthermore, isogeneic intrasplenic transplants of both adult rat fat and adrenal gland also failed to demonstrate adipocyte proliferation. It would appear that the presence of both adipocytes and pancreatic endocrine cells, particularly B cells, are required for the proliferation of adipocytes at the graft site.

We studied the chromatographic profile and the distribution of glucagon-related peptides in the human gastrointestinal mucosa, using radioimmunoassays directed against the glucagon 6-15 and 19-29 sequences, and against the glicentin sequences 15-30 and 61-69, and a radioreceptor assay for glucagon. Very small amounts of glucagon-related peptides were found in the gastric mucosa, whereas at least four different components could be identified in the distal intestine. One component (mean concentration 130 pmol/g ileal mucosa) is similar to porcine glicentin for size and C-terminal extension, but differs from the glucagon part of the molecule in the N-terminal extension. A second component (mean concentration 131 pmol/g) is probably identical to porcine peak II enteroglucagon (glicentin 33-69), and a third component (7.9 pmol/g) seems to be identical with glucagon. A fourth component containing the glucagon sequence plus an N-terminal extension was also identified (1.7 pmol/g). Thus the human intestinal mucosa contains large amounts of peptides containing the glucagon sequence; at least one of these probably also possesses glucagon-like bioactivity. The proposed structures of the four components are consistent with the base sequence of the first half of the human glucagon gene.

We have suggested that the order of cellular vascular perfusion within the islet is important in the regulation of islet hormone secretion. Anatomically, the A and D cells appear to be randomly dispersed throughout the mantle. Although islet capillary blood flow is known to be from the B-cell core to the A- and D-cell mantle, it has not yet been established whether the cells of the mantle may influence one another vascularly. Rat pancreata were perfused in vitro anterogradely and retrogradely with or without glucagon antibody in order to determine the order of cellular perfusion and interaction between the A and D cells in the islet mantle. Anterograde infusion of glucagon antibody did not affect insulin secretion, but rapidly decreased somatostatin secretion -46 +/- 8%, (p less than 0.005). Retrograde infusion of glucagon antibody decreased insulin secretion (-27 +/- 8%, p less than 0.005) but had no effect upon somatostatin secretion. This study not only confirms a core to mantle islet perfusion but also establishes that the A cell precedes the D cell in the terms of vascular perfusion. Thus within the islet, vascular borne insulin regulates the release of glucagon, which in turn, regulates the release of somatostatin. Somatostatin is vascularly neutral owing to its downstream position in the sequence (B to A to D) of cellular perfusion.

Insulin, proinsulin and glucagon extracted from lean rat pancreases were studied in radioimmunoassay, radioreceptorassay and bioassay systems. Extracted insulin behaved identically to a rat insulin used as a reference standard in radioimmunoassay. On the basis of its immunoreactivity, extracted insulin was slightly less potent (about 70%) than the rat standard insulin in competing with the binding of 125I-insulin to rat liver membranes (radioreceptorassay) and in stimulating glucose oxidation by rat fat cells (bioassay). Extracted glucagon and a pork glucagon used as a reference standard were indistinguishable in two radioimmunoassay systems for glucagon, in competing with the binding of 125I-glucagon to rat liver membranes (radioreceptorassay) and in stimulating adenylate cyclase in rat liver membranes (bioassay). Genetically obese rats (Zucker, "fatty") were compared to their lean littermates with respect to insulin, proinsulin and glucagon extracted from their pancreases. Proinsulin represented the same proportion of total immunoreactive insulin in both types of rats. In the radioimmunoassays, the radioreceptorassays and the bioassays, insulin, proinsulin and glucagon from obese rats were indistinguishable from insulin, proinsulin and glucagon from lean rats. It is concluded that the pancreatic hormones of obese ("fatty") rats possess the same immunoreactivity and biological potency as those of nonobese rats. This excludes the possibility that some alteration in the biological properties of pancreas insulin and/or glucagon of fatty rats could explain the metabolic abnormalities observed in this type of obesity.