The Unique Processing of
Glucagon in the Intestine in Traumatized Patients

Katsuhisa Tanjoh, MD, PhD

Ryouichi Tomita, MD, PhD

Nariyuki Hayashi, MD, PhD

 

Department of Emergency and Critical Care Medicine

Nihon University School of Medicine

30-1 Ohyaguchi Kamimachi Itabashi-ku

Tokyo 173-8610

Japan

 

KEY WORDS: Glucagon, trauma, immunoreactivity, peptides, kinetics,
intestine


ABSTRACT

Purpose: The kinetics of the pancreatic hormone glucagon in traumatized patients has not been minutely investigated as well as that of insulin, despite its significant influence on energy metabolism. 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. In addition, we discuss glucagon processes in the pancreas and intestine in traumatized patients in the early operative days.

Methods: Ten traumatized patients who had undergone emergency surgery were enrolled in this study (group S). Ten healthy volunteers were also enrolled as normal control subjects (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 the 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 high compared with that of group C (P < 0.05). Serum GLI was not significantly different between both groups. In all 10 patients in group S, a peculiar glicentin-like peptide (GLLP: MW approximately 8000 Da) other than pancreatic glucagon was seen on gel filtration chromatography, which was not seen in group C.

Conclusions: The kinetics and processing of glucagon in traumatized patients was different from those of healthy subjects. In traumatized patients, the peculiar processing of glucagon was processed in the intestine, which is different from the ordinary glucagon processing either in the pancreas or the intestine, generating the peculiar glicentin-like peptide (GLLP).

INTRODUCTION

Energy metabolism is markedly changed in traumatized and/or surgically stressed patients compared with that of healthy subjects. However, the endocrine system, especially the pancreatic hormones insulin and glucagon, greatly influence energy metabolism in insulted patients,1-3 although the kinetics of glucagon have not been investigated as thoroughly as that of insulin in insulted conditions. The interpretation of the results of glucagon levels in serum has been complicated because of the existence of various glucagon-related peptides such as preproglucagon, glicentin, and oxyntomodulin generated not only by the pancreas, but also other organs, especially the intestines.4 All of these glucagon-related peptides detected in serum by radioimmunoassays are precursors of pancreatic glucagon. In this study, we investigated serum glucagon using the glucagon-specific C-terminal radioimmunoassay (RIA) and other glucagon-related peptides levels using nonspecific N-terminal RIA in traumatized patients who underwent emergency surgery. The molecular forms of these glucagon and glucagon-related peptides were also estimated using the gel filtration chromatography method to make the serum results clear, which the respective glucagon-related peptide described above generated. Furthermore, we discussed the glucagon processes in these traumatized patients according to those findings to make clear the influence of the insult to the kinetics of glucagon.

PATIENTS

This study was performed in the Department of Emergency and Critical Care Medicine, Nihon University Itabashi Hospital, Tokyo, Japan, in accordance with the principles established in Helsinki and with the approval of the Institutional Research Review Committee at Nihon University Itabashi Hospital. Informed consent was obtained from each patient before the study. Ten traumatized patients undergoing emergency surgical treatment were enrolled in this study (group S; Table 1). Six patients were males and four were females, between ages 27 and 62 years with (mean ± standard deviation [SD], 45.0 ± 12.1 years). Injured organs, injury severity score (ISS),5 APACHE (Acute Physiology and Chronic Health Evaluation) II score6 on admission, surgical procedures undergone, and clinical outcomes are shown in Table 1. The APACHE II score of all patients in group S indicated over 12 points on admission. Ten healthy volunteers, 5 males and 5 females, with a mean ± SD age of 41.4 ± 11.3 years, were also enrolled in this study as the control group (group C).

METHODS

Blood samples were collected from the antecubital vein, in the early morning in the fasting state in group C and on the second postoperative day in group S, into ice-chilled tubes containing 500 kilo-international units of aprotinin (Trasylol; Bayer Leverkusen) and 1.2 mg of EDTA per milliliter of blood. The samples were centrifuged immediately and the plasma was separated and kept at -20˚C until assayed.

Glucagon Specific C-terminal Radioimmunoassay (Immunoreactive Glucagon) Using Antisera OAL-1237
and Nonspecific N-terminal RIA (Glucagon-Related Immunoreactivity) Using Antisera OAL-196

The antiserum OAL-123 reacts with the C-terminal 24-29 sequence of glucagon and detects the glucagon sequence on the RIA specifically, because the C-terminal extended sequence, the hexapeptides in larger molecular precursors of glucagon (glucagon-related peptides) such as preproglucagon, glicentin, and oxyntomodulin, participate in the masking of the C-terminal immunodeterminant of the glucagon portion of these precursors4 (Figure 1). The antiserum OAL-196 reacts with the N-terminal 1-26 sequence of glucagon and detects all of these glucagon-related peptides, including glucagon, on the RIA (Figure 1).

Glucagon was radioiodinated using the Chloramine T method9 and the labeled compound was purified using anion-exchange chromatography on a Sephadex A-25 column. Specific activity of the purified 125I-labeled glucagon ranged from 510 to 620 Ci/g, depending on preparations. The standard diluent used for the assay was phosphate buffer (40 mmol/L, pH 7.4) containing, per liter, 2.0 g of bovine serum albumin, 7.5 mmol of EDTA, and 75 mg of sodium azide. Mixed in each incubation tube were 200 µL of diluted antiserum (OAL-1237/OAL-1968 diluted 60,000-fold for each), 200 µL of labeled glucagon, 200 µL of glucagon standard or unknown sample, and 200 µL of standard diluent containing 1000 kilo-international units of aprotinin. The mixture was incubated for 72 hours at 4˚C. Free and bound 125I-labeled glucagon were separated using the dextran-coated charcoal method. Immediately before dextran-coated charcoal was added, 200 µL of normal sheep serum was added to the tubes containing standard glucagon and 200 µL of standard diluent to the tubes of plasma samples. After incubation for 1 hour at 4˚C, the suspension was centrifuged (2000 g, 20 min, 4˚C) and the supernatant was decanted. The radioactivity in both the supernatant fluid and the precipitate was counted using a gamma-spectrometer.

Evaluations of the Molecular Forms of Glucagons in Serum Using Gel
Filtration Chromatography

Gel filtration chromatography for evaluating the molecular forms of the assessed glucagon-related peptides (immunoreactive glucagon [IRG] and glucagon-related immunoreactivity [GLI]) in serum were performed.7,8 The sera obtained from patients in both groups were fractionated by gel filtration. Each blood sample was centrifuged immediately to separate the serum and then treated with 95% ethanol and applied to a 1.5 ¥ 91 cm Sephadex G
column equilibrated with phosphate buffer (40 mmol/L, pH 7.4) containing, per liter, 2.0 g of bovine serum albumin, 7.5 mmol of EDTA, and 0.1 mol of NaCl. The flow rate was 5 mL/h and the fraction volume was 2.6 mL each. The column was preincubated with Blue Dextran (Pharmacia Fine Chemicals, Sweden), 125I-labeled bovine serum albumin, 125I-labeled glicentin, and 125I-labeled glucagon as the molecular markers. Filtrated materials were assayed using both glucagon-specific C-terminal (IRG) and nonspecific N-terminal (GLI) RIA as described previously in this article.7,8 The tests described previously were performed in one patient in each group using duplicate assessments.

Statistical Analysis

Results of the glucagon RIAs in serum were expressed as means ± SD. Statistical analysis was performed using Student's paired t test (two-tailed), and P values of less than 0.05 were considered significant.

RESULTS

Glucagon-Specific C-terminal Radioimmunoassay (Immunoreactive Glucagon: IRG) and Nonspecific
N-terminal RIA (Glucagon-Related Immunoreactivity: GLI)

Serum IRG in group S (114.7 ± 35.4 pg/mL) was significantly high compared with that of group C (81.4 ± 21.4 pg/mL; P < 0.05). Serum GLI in group S (310.8 ± 177.3 pg/mL) was slightly high compared with that of group C (293.8 ± 188.2 pg/mL), but not significantly different (see Figure 2).

Gel Filtration Chromatography of IRG and GLI

1) Group C (Figure 3): At the fasting stage in all subjects in group C, GLI showed four peaks corresponding to the molecular weights of bovine serum albumin (BSA), glicentin, oxyntomodulin, and pancreatic glucagon. In contrast, IRG showed only one peak corresponding to the molecular weight of glucagon.

2) Group S (Figure 4): During fasting in all subjects in group S, GLIs showed four peaks corresponding to the molecular weights of BSA, glicentin, oxyntomodulin, and glucagons, which were similar to those of group C. Whereas IRG showed two characteristically different peaks, one had a similar but slightly lower molecular weight of that of glicentin (glicentin-like peak [GLLP]), which was not seen in normal subjects and the other peak corresponding to pancreatic glucagon in molecular weight.

DISCUSSION

Although the pancreatic hormone glucagon possesses various biologic activities second in importance to that of insulin and plays a major role in carbohydrate, amino acid, and lipid metabolism, little is known about the kinetics of glucagon under traumatized or surgically stressed conditions, in contrast to the kinetics of insulin which have been thoroughly investigated. Glucagon and glucagon-related peptides, ie, glucagon precursors such as glicentin, oxyntomodulin, and preproglucagon, were not only produced in A cells in the pancreas, but also in other organs, especially L cells in the intestines.4 The existence of these various glucagon-related peptides in serum complicate the interpretation of the assessed results of the kinetics of glucagons. In this study, we assessed the kinetics of serum glucagon and glucagon-related peptides using glucagon-specific C-terminal and nonspecific N-terminal RIA.7,8 In addition, the molecular forms of these peptides were assessed using gel filtration chromatography to evaluate the kinetics of glucagon and glucagon-related peptides in traumatized patients. However, serum GLI (which reacts with OAL196, nonspecific glucagons N-terminal RIA) in group S was not significantly increased; IRG (which reacts with OAL123, specific glucagon C-terminal RIA) in group S was significantly higher than those in group C, probably affected by the stress stimulation in the traumatized and surgically stressed patients.

In the present study, we also evaluated the molecular form of the IRGs and GLIs using gel filtration chromatography to clarify the kinetics of glucagons in the insulted status.

In group C (Figure 3), during fasting, we found four peaks of peptides corresponding to the molecular weights of bovine serum albumin, glicentin, oxyntomodulin, and pancreatic glucagon using N-terminal nonspecific glucagon RIA and only one peak corresponding to the molecular weight of pancreatic glucagon using C-terminal-specific glucagon RIA. These glucagon and glucagon-related peptides are possibly released mainly from the pancreas and small intestines in humans.9,10 Although both of these pancreatic and intestinal glucagons were suggested to be generated from the same precursor, preproglucagon, the final peptides produced, are different in these two organs because of the different glucagon processing between A cells in the pancreas and L cells in the small intestine, ie, oxyntomodulin and pancreatic glucagon are mainly produced in the pancreas and glicentin and also oxyntomodulin are produced in the small intestines.4,12,13 The peak corresponding to the molecular weights of bovine serum albumin, measured in the present study in gel filtration chromatography in nonspecific N-terminal assay, are suggested to be the precursor of pancreatic glucagon, preproglucagon.

In group S (Figure 4), GLI showed four peaks which were almost similar to those in group C, corresponding to the molecular weights of bovine serum albumin, glicentin, oxyntomodulin, and pancreatic glucagon, respectively. However, there were two different peaks observed in the C-terminal assay, one had a similar but slightly lower molecular weight than that of glicentin (GLLP) and the other corresponded to pancreatic glucagon in molecular weight. This GLLP was not seen in group C and was also observed in totally pancreatectomized patients as previously reported.14,15 Considering these findings, this characteristic GLLP is suggested to be released from the intestines and to be markedly different from glicentin itself, because the OAL-123 antibody used in the present study for the glucagon C-terminal RIA does not clearly react with it.7 The other peak corresponding to the molecular weight of pancreatic glucagon was similar to that in group C. This suggests that the increased IRG assessed in group S must consist of the occurrence of this GLLP and the ordinary pancreatic glucagon. This characteristic GLLP is suggested to be the glicentin fragment 1-62 dissociated from the C-terminal fragment 64-69 (hexapeptide) from glicentin, based on the findings in this study that this peptide had a slightly lower molecular weight than glicentin as observed on gel filtration chromatography and reacted with glucagon C-terminal antisera OAL-123. The suggestion that the peculiar glucagon processing generating GLLP was presumably carried out in the intestine, not in the pancreas, was supported by the present findings that the dissociation portion of glicentin molecule for the generation of GLLP is a specific processing portion in the intestines4 and furthermore, by the findings that GLLP was also seen in totally pancreatectomized patients as previously reported.14,15

This suggests that the glucagon-related peptides originated from the preproglucagon16,17 in the pancreas and the intestines and processed as shown in Figure 5 in traumatized and surgically stressed patients, based on the present findings. At first, the signal peptide in the N-terminal was dissociated in both the pancreas and the intestines16 following the glucagon-like peptide in the C-terminal in the intestine; and the glicentin-related pancreatic peptide (glicentin 1-31 fragment) in the N-terminal in the pancreas16 were dissociated from the preproglucagon in succession, similar to the ordinary glucagon processing, generating pancreatic glucagon molecule in the pancreas and glicentin molecule in the intestines. In normal subjects, the majority of the generated glicentin molecule in the intestines is further dissociated by the GRPP, generating a oxyntomodulin molecule. However, glicentin in traumatized and surgically stressed patients can be dissociated by the glicentin 64-69 fragment, which is significantly different from the ordinary glucagon processing either in the pancreas or the intestines, generating the peculiar GLLP. This suggests that the GLLP observed in group S in glucagon-specific C-terminal assays in the present study were generated from preproglucagon by the unique glucagon processing in the intestines in a markedly different manner from the typical glucagon processing described previously in this article.

Although the reason why this unique glucagon processing occurred in traumatized and surgically stressed patients is currently unknown, it could be presumed to be associated with changes in endocrine circumstances, especially the impaired glucose tolerance that is common to both traumatized patients and patients after pancreatectomy as previously reported.14,15,18 Therefore, the peculiar glucagon processing for generating GLLP could be a hormonal adaptation mechanism related to the extraordinary endocrine or stressed circumstances. Further investigations are needed to clarify the processing of glucagon in traumatized patients.

CONCLUSION

The kinetics and processing of glucagon in traumatized patients was different from those of normal subjects. The peculiar processing of glucagon observed in these patients was probably processed in the intestines, generating the peculiar glicentin-like peptides (GLLP), which might be the result of the changes in the endocrine circumstances.

References

1. Unger RH: Glucagon and the insulin: Glucagon ratio in diabetes and other catabolic illness. Diabetes 20:834-838, 1971.

2. Unger RH: Insulin-glucagon ratio. Isr J Med Sci 8:252-257, 1972.

3. Parrilla R, Goodmn MN, Toews CJ: Effect of glucagon: Insulin ratio on hepatic metabolism. Diabetes 23:725-731, 1974.

4. Yanaihara N, Yanaihara CH, Nishida T, et al: Synthesis of glicentin- and proglucagon-related peptides and their immunological properties. In: Rosselin G, Formagoet P, Bonifile S, eds. Hormone Receptors in Digestion and Nutrition. Amsterdam: Elsevier; 1979, pp 65-68.

5. Baker SP, O'Neill B, Haddon W, et al: The injury severity score: A method for describing patients with multiple injuries and evaluating emergency care. J Trauma 14:187-196, 1974.

6. Knaus WA, Draper EA, Wagner DP, Zimmerman JE: APACHE II: A severity of disease classification. Crit Care Med 13:818-829, 1985.

7. Nishino T, Kodaira T, Shin S, et al: Glucagon radioimmunoassay with use of antiserum to glucagon C-terminal fragment. Clin Chem 27:1690-1697, 1981.

8. Nishino T, Kodaira T, Shin S, et al: Production of antisera to des Asn28 Thr29 Homoser27-glucagon; The development of radioimmunoassay for total glucagon-like immunoreactivity in human plasma. Endocrinol Japon 28:419-427, 1981.

9. Hunter WM, Greenwood FC: Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194:495-496, 1962.

10. Holst JJ, Pedersen JH, Baldissera F: Circulating glucagon after total pancreatectomy in man. Diabet 25:396-399, 1983.

11. Bajorunas DR, Fortner JG, Jaspan JB: Glucagon immunoreactivity and chromatographic profiles in pancreatectomized humans. Diabet 35:886-893, 1986.

12. Tager HS, Steiner DF: Isolation of glucagon-containing peptide: Primary structure of a possible fragment of proglucagon. Proc Natl Acad Sci USA 70:2321-2325, 1973.

13. Yanaihara N, Yanaihara CH, Sakagami M, Mochizuki T, Kubota M: Hormone synthesis. In: Bloom SR, Polak JM, eds. Gut Hormones, 2nd ed. Edinburgh: Churchill Livingstone; 1981, pp 25-31.

14. Tanjoh K, Shima A, Aida M, et al: Kinetics of glucagon like peptides in postpancreatectomy patients [in Japanese]. Gut Hormones 13:213-218, 1995.

15. Tanjoh K, Ryouichi T, Masahiro Fukuzawa, Nariyuki Hayashi: Peculiar glucagon processing in the intestine is the genesis of the paradoxical rise of serum pancreatic glucagon in patients after total pancreatectomy. Hepato-Gastroenterology 50:535-540, 2003.

16. Lopez LC, Frazier ML, Su CJ, Kumar A, Saunders GF: Mammalian pancreatic preproglucagon contains three glucagon related peptides. Proc Natl Acad Sci USA 80:5485-5489, 1983.

17. Bell GI: Hamster preproglucagon contains the sequence of glucagon and two related peptides. Nature 302:716-718, 1983.

18. Tanjoh K, Tomita R, Hayashi N: The peculiar processing of glucagon and glucagon-related peptides in patients after pancreatectomy. Hepato-Gastroenterology 49:825-832, 2002.

 

Figure 1. The sequence of glucagon-related peptides and the immunodeterminant portion of antisera used on the glucagon radioimmunoassays (RIAs) in this study. The antiserum OAL-123 used on the glucagon-specific C-terminal RIA reacts with the C-terminal 24-29 sequence of glucagon and detects the glucagon sequence on the RIA specifically. The antiserum OAL-196 used on the glucagon nonspecific N-terminal RIA reacts with the N-terminal 1-26 sequence of glucagon and detects not only glucagon, but also other glucagon-related peptides on the RIA.

 

 

Figure 2. Values of the serum glucagon immunoreactivities (GLI) and immunoreactive glucagons (IRG) in group C and group S: There are no significant differences in the values of serum GLI among groups S and C. Serum IRG in group S is significantly higher than that of group C.

 

Figure 3. Gel filtration chromatography of group C: GLIs showed four peaks corresponding to the molecular weights of preproglucagon, glicentin, oxyntomodulin, and pancreatic glucagon. In contrast, IRGs showed only one peak corresponding to the molecular weight of pancreatic glucagon.

 

Figure 4. GLI showed four peaks, similar to group C. IRG showed two characteristically different peaks, one having a similar but slightly lower molecular weight than that of glicentin (glicentin-like peak [GLLP]) and the other corresponding to pancreatic glucagon in molecular weight.