Changes in glucagon processing occurring in the intestines of surgically stressed patients

BACKGROUND/AIMS: The kinetics of the pancreatic hormone glucagon in surgically stressed patients has not been investigated as thoroughly as that of insulin, despite its significant influence on energy metabolism in surgically stressed conditions. In the present study, we examined the kinetics of glucagon and glucagon-related peptides assessed by radioimmunoassay, and the molecular forms of these peptides using gel filtration chromatography, and in addition discuss glucagon processes in the pancreas and intestine in surgically stressed patients. METHODOLOGY: Ten patients who had undergone abdominal surgery for acute abdominal disorders were enrolled in this study (group S). Ten healthy volunteers were also enrolled as normal controls (group C). The serum level of glucagon and glucagon-related peptides were assessed in the early morning fasting state in both groups, on the second postoperative day in group S, using glucagon nonspecific N-terminal (glucagon-like immunoreactivity: GLI) and specific C-terminal (immunoreactive glucagon: IRG) radioimmunoassays. The molecular forms of these peptides were also estimated using the gel filtration chromatography method. RESULTS: Serum IRG in group S was significantly higher than that of group C (P < 0.05). Serum GLI was not significantly different between the two groups. In all patients except one in group S, a peculiar glicentin-like peptide (GLLP: MW about 8000) other than pancreatic glucagon was seen in gel filtration chromatography, which was not seen in group C. CONCLUSIONS: The kinetics and processing of glucagon in surgically stressed patients were quite different from those of healthy subjects. In surgically stressed patients, peculiar processing of glucagon occurred in the intestine, which was quite different from ordinary glucagon processing either in the pancreas or the intestine, generating GLLP.     

Peculiar glucagon processing in the intestine is the genesis of the paradoxical rise of serum pancreatic glucagon in patients after total pancreatectomy

BACKGROUND/AIMS: Although glucagon has been detected even in the serum of totally pancreatectomized patients and the origin was suggested to be the intestine, the kinetics of glucagon are not well known after pancreatectomy. In the present study, we investigated the kinetics of glucagon and glucagon-related peptides in pancreatectomy patients and discuss the glucagon processes. METHODOLOGY: Ten patients who had undergone total pancreatoduodenectomy reconstruction using Billroth II type procedures (group PX) and 12 normal subjects (group C) were also enrolled in this study. All patients received a 75-g oral glucose tolerance test in the early morning fasting state. Serum glucagon levels were assessed using the glucagon specific C-terminal (immunoreactive glucagon: IRG) and nonspecific N-terminal (glucagon-like immunoreactivity: GLI) radioimmunoassays. The molecular forms of these glucagon-related peptides were also estimated using the gel filtration chromatography method before and after the oral glucose load. RESULTS: After the glucose load, serum GLIs were increased significantly in group PX suggesting that these were affected by the alimentary tract reconstructions. Serum IRGs were significantly increased in group PX, but decreased in group C after oral glucose load suggesting that these paradoxical increased responses in group PX might be associated with the insulin secretion deficiencies, but not associated with the alimentary tract reconstruction. CONCLUSIONS: The paradoxical rise in IRGs based on the findings of gel filtration chromatography in group PX were possibly due to the generated peculiar glicentin-like peptide from the glucagon precursor, preproglucagon, after total pancreatectomy, which might be processed in intestines in association with the insulin deficiencies.     

The peculiar processing of glucagon and glucagon-related peptides in patients after pancreatectomy

BACKGROUND/AIMS: Although, glucagon has been detected even in the serum of totally pancreatectomized patients and the origin was suggested to be the intestine, the kinetics of glucagon is not well known after pancreatectomy. In this study, we investigated the kinetics of glucagon and glucagon-related peptides and discuss the glucagon processes in the pancreas and intestine in pancreatectomy patients. METHODOLOGY: Seven patients who had undergone pancreatoduodenectomy reconstruction using Child's procedures (distal pancreatojejunostomy, choledochojejunostomy and gastrojejunostomy) (group PD) and five patients who had undergone distal pancreatectomy (group DP) served as the subjects of this study. In addition to these two groups, five patients who had undergone gastrectomy reconstructions using the Billroth II procedure (group GX), to examine whether the alimentary tract reconstructions themselves would have any effect on the kinetics of glucagon, and 10 normal subjects (group C) were also enrolled in this study. All patients received a 75-g oral glucose tolerance test in the early morning fasting state. Serum glucagon levels were assessed using the glucagon non-specific N-terminal (glucagon-like immunoreactivity: GLI) and specific C-terminal (immunoreactive glucagon: IRG) radioimmunoassays. The molecular forms of these glucagon-related peptides were also estimated using the gel filtration chromatography method before and after the 75-g oral glucose load. RESULTS: After the glucose load, serum GLIs were increased significantly in groups GX and PD suggesting that these were affected by the alimentary tract reconstructions. Serum IRGs including true pancreatic glucagon were slightly increased in groups PD and DP after oral glucose load suggesting that these paradoxical responses might be associated with the glucose tolerance deficiencies observed in both groups, but not associated with the alimentary tract reconstruction. CONCLUSIONS: The paradoxical rise of IRGs based on the findings of gel filtration chromatography were possibly due to the generated peculiar glicentin-like peptide and pancreatic glucagon from the glucagon precursor, preproglucagon, after pancreatectomy, which is processed in association with the glucose tolerance deficiencies after pancreatectomy.     

Fetal rat intestinal cells in monolayer culture: a new in vitro system to study the glucagon-like immunoreactive peptides

To establish an in vitro model to investigate the glucagon-related peptides, fetal rat intestinal cells were enzymatically dispersed and placed into culture for up to 7 days. After 1 day in culture, the presence of epithelial-like cells containing glucagon-like immunoreactivity (GLI) was demonstrated using immunocytochemical techniques. The cell peptides were extracted by passage through a cartridge of octadecylsilyl silica and characterized by gel filtration and RIA. Two GLI moieties were detected with apparent mol wts of 11,000-12,000 and 5,000-6,000. The immunoreactive profile obtained for the cells in culture was identical to that of both whole fetal rat intestine and adult rat ileum. The presence of glucagon could not be demonstrated in any of the extracts. The basal levels of GLI and apparent immunoreactive glucagon (IRGa) were 1,457 +/- 381 and 198 +/- 57 pg/dish, respectively, on day 1 of culture. The GLI content of the cells, but not the IRGa, declined with time in culture for up to 5-7 days (P less than 0.03). Addition of insulin to the culture medium (10 or 100 mU/ml) did not influence the decrease in GLI content of the cells, but did inhibit the production of IRGa (P less than 0.05). Addition of 500 mg/dl glucose to the cells in the presence of 20 microU/ml insulin increased the secretion of GLI by 42 +/- 7% over 2 h (P less than 0.05). The stimulation by glucose was not seen in the absence of insulin or with higher insulin concentrations (100 microU/ml), nor did insulin alone (100 microU/ml) have any effect on the release of GLI. Thus, fetal rat intestinal cells in culture produce the GLI peptides, and secrete them in response to glucose. This system may provide a means by which the synthesis and control of secretion of the glucagon-related peptides can be investigated.     

Mouse salivary glands secrete a glucagon-degrading enzyme, not glucagon

Salivary glands have been reported to synthetize glucagon in various animal species. We therefore studied the glucagon-like immunoreactivity (GLI) in mouse saliva. Stimulation with phenylephrine evoked a 15-fold increase of salivary GLI output. On Sephadex G-50 gel filtration, the salivary GLI was significantly larger than glicentin, the hitherto largest known glucagon-related peptide; furthermore, the immunoreactivity was not absorbable on glucagon immunoadsorbent. 125I-Labeled glucagon incubated with high GLI containing saliva, and subjected to gel filtration and immunoadsorption was degraded to low molecular weight, nonimmunoreactive moieties. Among EDTA, phenylmethylsulfonylfluoride, Pepstatin-A, and N-ethylmaleimide, only N-ethylmaleimide inhibited the degradation. Renin, also found in mouse saliva, degraded the tracer but did not cochromatograph with salivary GLI. In conclusion, GLI of mouse saliva is not a peptide containing a glucagon-immunoreactive sequence but represents tracer-degrading activity, probably composed of sulfhydryl enzymes.     

Inhibition of exaggerated gastrointestinal glucagon responses in chronic pancreatitis by somatostatin

Oral glucose administration caused an exaggerated release of cross-reacting gastrointestinal glucagon-like immunoreactivity (GLI) and a slight early rise in immunoreactive glucagon (IRG) concentration in patients with chronic pancreatitis, who have impaired insulin release. Intravenous administration of 200 microgram of somatostatin, followed by infusion of 200 microgram over 2 1/2 h, abolished the GLI and insulin responses, but did not change glucose tolerance. This contrasts with the relatively minor effects of somatostatin on GLI responses in control subjects where the clear deterioration in glucose tolerance may relate to inhibition of insulin release.     

Plasma levels of glucagon-like polypeptides in patients with esophagoplasty

Seven patients with interposition of a jejunal tract between the esophagus and stomach or duodenum (EP group) and ten healthy normal volunteers have been submitted to an oral glucose load (OGTT) to clarify the significance of glucagon-like polypeptides (GLI) induced by glucose in carbohydrate metabolism. Blood glucose (BG) and GLI plasma levels were significantly higher in the EP group than in the normal one (p less than 0.01), respectively 30, 60, 90 min for BG and during all the test for GLI. The fasting immunoreactive glucagon (IRG) plasma levels were significantly lower in patients than in normals (p less than 0.05) whereas a marked and significant increase was observed in the EP group (p less than 0.01) from 90 to 240 min. The precocious stimulation of jejunal mucosa and the rapid intestinal transit which occur in these patients may explain the elevated GLI (probably glicentin) plasma levels. The rise in IRG plasma levels might be due to the enzymatic transformation of glicentin. The role of these types of glucagon on carbohydrate metabolism is still to be fully clarified.     

Chemical characterization of persistent immunoreactive glucagon in eviscerated rats

Summary Extraction of rat submaxillary salivary glands and ileal mucosa yielded a single immunoreactive glucagon component of 25,000 daltons and a major peak of approximately 9,000 daltons respectively. Eviscerated rats with and without functional livers were found to have decreased, but persistent levels of immunoreactive glucagon and total immunoreactive glucagon, as measured with a pancreatic specific and cross reacting antiserum respectively. Gel filtration of serum samples on Sephadex G-50 fine and G-200 superfine demonstrated that there was no measurable 3485, 9000 or 25,000 dalton glucagon immunoreactivity. The persistent immunoreactive glucagon in the serum of eviscerated rats is a heterogeneous mixture of nonspecific materials and does not contain submaxillary or intestinal glucagon-related materials. These findings suggest caution in the interpretation of glucagon immunoreactivity in serum and demonstrate the necessity for chromatographic analysis of circulating IRG.     

Glucagon-like immunoreactive peptides in a rat ileal epithelial cell line (IEC-18)

The presence of cells containing glucagon-like immunoreactive (GLI) peptides was demonstrated in a rat ileal epithelial cell line (IEC-18) by both immunofluorescence and radioimmunoassay. When cell extracts were subjected to gel filtration chromatography, the cells were found to contain 3.5 Kd glucagon in addition to significant quantities of large molecular weight GLI peptides (apparent molecular weights of 4, 6, 8 and 10 Kd) and a 9 Kd peptide with apparent glucagon immunoreactivity. This was in contrast to extracts of adult rat ileum, which contained only large molecular weight GLI peptides (apparent molecular weights of 6 and 12 Kd). Production of GLI peptides by the IEC-18 cells was stimulated by glucose (p less than 0.02) and inhibited by insulin (p less than 0.01). In conclusion, these results demonstrate that the IEC-18 cells produce both GLI peptides and glucagon, and thus support the notion that proglucagon processing is cell-specific. IEC-18 cells may therefore provide a tool for investigations of some aspects of GLI peptide and glucagon synthesis.     

Secretion of immunoreactive insulin and glucagon in hamsters bearing a transplantable insuloma.

Glucose, insulin (IRI), pancreatic (IRG) and total (GLI) immunoreactive glucagon were measured in the serum of normal hamsters and of hamsters with an insulin- and glucagon-secreting, transplantable insuloma. The tumor-bearing animals were hypoglycemic, hyperinsulinemic and hyperglucagonemic. The pancreatic islets of tumor-bearing animals secreted less glucagon and insulin in response to arginine or to changes in the glucose concentration of the medium, than did the islets of control hamsters. In addition, the introduction of glucose into the gastro-intestinal tract, which caused a significant rise in the serum GLI concentration of normal hamsters, failed to do so in the tumor-bearing animals. The results suggest that the high levels of serum glucagon and insulin induced by the tumor, suppressed IRI, IRG and GLI secretion in these animals.     

Identification and localization of glucagon-related peptides in rat brain.

Immunochemical and immunocytochemical techniques have been used to identify and characterize glucagon-related peptides of the rat central nervous system. These peptides show immunoreactivity with antiglucagon sera directed towards the central portion of the hormone, but not with antisera specific for the free COOH terminus of glucagon. Highest concentrations were found in hypothalamus (6.1 +/- 1.6 ng/g wet weight) although lower amounts (approximately 2 ng/g) were found in cortex, thalamus, cerebellum, and brain stem. Gel filtration of brain extracts revealed at least two immunoreactive forms, which have molecular weights of about 12,000 and 8000. Both peptides had radioimmunoassay dilution curves parallel to the curve for glucagon and both had identical counterparts in extracts of rat intestine. Digestion of the brain and intestinal peptides with trypsin plus carboxypeptidase B released the immunoreactive COOH-terminal tryptic fragment of pancreatic glucagon from these larger forms. Immunocytochemical studies using antiglucagon serum and peroxidase-antiperoxidase staining identified glucagon-like material in neuronal cell bodie and processes in the magnocellular portion of the paraventricular nucleus, as well as in scattered cells in the supraoptic nucleus and in fibers in the median eminence. These results suggest that glucagon-containing peptides that have undergone the intestinal type of posttranslational modification are present in neuronal cells of the rat hypothalamus.     

Glucagon-related peptides in the human gastrointestinal mucosa

Summary 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.     

Plasma immunoreactive glucagon fractions in four cases of glucagonoma: Increased “Large glucagon — immunoreactivity”

Summary 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 (>20000 mol. wt.), peak II (primarily 9000 mol. wt.), peak III pancreatic glucagon (3500 mol. wt.) and peak IV small glucagon (<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 porcine pancreatic glucagon. The presence of heterogeneous 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.Supported in part by NIH Grant #AM 17431-02.     

Gastric operations and glucose homeostasis. II. glucagon and secretin.

Alimentary hyperglycemia in patients who have undergone gastric operations may be due, in part, to altered intestinal signals for glucose disposition. We measured glucose, immunoreactive insulin (IRI) pancreatic glucagon (IRG), and glucagon-like immunoreactivity (GLI) after oral glucose in patients with prior antrectomy or vagotomy and pyloroplasty and in normal individuals. All subjects had normal assimilation coefficients for intravenous glucose, which suggests that the responsiveness of the pancreatic beta-cells had not been altered by the surgical procedures. The early hyperglycemic response to oral glucose and the associated elevation of plasma GLI were much greater and the IRI levels slightly higher in both experimental groups in comparison to normal subjects. A decrease in the level of IRG, albeit not statistically significant, was noted in all groups after the ingestion of glucose. In gastrectomy patients, secretin infusion during repeated oral glucose tolerance tests partially corrected the hyperglycemia and lowered plasma GLI and IRI levels. The responses of the vagotomy and pyloroplasty patients and of the normal subjects were not altered by secretin infusion. We conclude that the intolerance or oral glucose after gastric surgery may be related to elevated GLI levels, and that the beneficial effect of secretin may be due to its ability to decrease these levels.     

Oxyntomodulin and glicentin: brain-gut peptides in the rat

Glucagon-like materials and glucagon have been identified by immunoassay and immunocytochemistry in the mammalian central nervous system. However, the molecular forms relevant to brain glucagon-like immunoreactivity (GLI) have not been precisely defined. In the rat small intestine, more than 90% of GLI is constituted by two peptides: oxyntomodulin (OXM) and glicentin. This work was initiated to characterize and determine the concentrations of these two peptides and glucagon in the rat central nervous system and to compare their relative proportions with those found in the gut. Different regions from the adult rat brain were analyzed by HPLC in association with RIA, using a central glucagon antiserum and an antibody directed toward the C-terminal end of OXM and glicentin. The elution profiles of hypothalamus extracts were constituted by two main peaks, both detected by the two antibodies used and displaying the same retention times as glicentin and OXM, respectively. A third small peak, which coeluted with glucagon, was constantly recorded with the central glucagon antiserum. The percentages of glicentin, OXM, and glucagon in 10 hypothalami were 37 +/- 1%, 55 +/- 1%, and 8 +/- 2%, respectively (n = 8). This distribution was quite similar to that in small intestinal extracts (38 +/- 1%, 59 +/- 1%, and 1.3 +/- 0.1%, respectively; n = 7); however, the peptide concentrations were almost 50-fold greater in intestine than in hypothalamus. In the medulla oblongata, the same peptide ratio was observed, with 10-fold lower concentrations compared to those in hypothalamus. In olfactory bulb, cerebellum, and cortex the concentrations were close the the detection limit, whereas they could be not detected in the pituitary. The combination of HPLC and specific RIAs allowed us to unambiguously characterize OXM and glicentin as the major components of GLI in the rat hypothalamus and medulla oblongata. The same proportion of these two peptides in the central nervous system and the gut indicates that a similar posttranslational processing exists in these rat tissues, another example of the brain-gut axis.     

Glicentin 1–61 probably represents a major fraction of glucagon-related peptides in plasma of anaesthetized uraemic pigs

Summary 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 (M_r) components comprising glicentin and moieties of slightly lower M_r 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 M_r glucagon-related peptides found in uraemia in pigs.