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ORIGINAL ARTICLE Table of Contents   
Year : 2013  |  Volume : 10  |  Issue : 2  |  Page : 91-94
The comparison of the intestinal adaptation effects of subcutaneous and oral insulin in a rats with short bowel syndrome


1 Department of Pediatric Surgery, Ondokuz Mayis University, Samsun, Turkey
2 Department of Biochemistry, Ondokuz Mayis University, Samsun, Turkey
3 Department of Pathology, Ondokuz Mayis University, Samsun, Turkey

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Date of Web Publication15-Jul-2013
 

   Abstract 

Aim: Insulin has been reported to have positive effects on intestinal adaptation after short bowel syndrome when applicated oral or subcutaneously. The purpose of this study is to compare the intestinal adaptation effects of subcutaneous and oral routes of insulin in rats with short bowel syndrome. Materials and Methods: The short bowel syndrome (SBS) was performed through 70-75% of small intestinal resection and an end-to-end anastomosis. The control group rats underwent SBS only. In the second group, oral insulin (1 U/ml) was administrated twice-daily. In the last group, the insulin was administrated subcutaneously (1 U/kg) as in the control group. All rats were killed on day 15. Outcome parameters were weight of small intestine, the crypt length, villous depth, the blood levels of vascular endothelial growth factor (VEGF), and granolocyt-monocyst colony-stimulating factor (GMCSF). Results: Intestinal weight was significantly more in oral insulin group and subcutaneous insulin group than in the control group (72.6 ± 4.3, 78.6 ± 4.8 and 59.7 ± 4.8) (P < 0.05). There was no difference between the groups according to villus length, crypt depth, and villous/crypt ratio both in proximal and distal parts of the resected bowel (P > 0.05). VEGF values were not statistically significant between the groups (200.3 ± 41.6, 178.9 ± 30.7 and 184.3 ± 52.2) (P > 0.05). GMCSF was statistically higher in the control group than in other groups (3.34 ± 1.34, 1.56 ± 0.44 and 1.56 ± 0.44) (P < 0.05). Conclusion: Insulin has positive effects on intestinal adaptation in short bowel syndrome. Subcutaneous administration is slightly more effective than the oral route.

Keywords: Insulin, intestinal adaptation, short bowel syndrome

How to cite this article:
Bicakci U, Tuncel OK, Bilgici B, Tander B, Ariturk E, Rizalar R, Alici O, Bernay F. The comparison of the intestinal adaptation effects of subcutaneous and oral insulin in a rats with short bowel syndrome. Afr J Paediatr Surg 2013;10:91-4

How to cite this URL:
Bicakci U, Tuncel OK, Bilgici B, Tander B, Ariturk E, Rizalar R, Alici O, Bernay F. The comparison of the intestinal adaptation effects of subcutaneous and oral insulin in a rats with short bowel syndrome. Afr J Paediatr Surg [serial online] 2013 [cited 2019 Nov 14];10:91-4. Available from: http://www.afrjpaedsurg.org/text.asp?2013/10/2/91/115030

   Introduction Top


Short bowel syndrome (SBS) is defined as massive intestinal loss with compromised bowel adaptation in congenital or acquired pathologies. [1] Patients with SBS have many complications related to prolonged total parenteral nutrition (TPN) and long-term hospitalization such as cholestasis, liver failure, multiple systemic infections, and central line complications. In SBS, intestinal adaptation is crucial for survival and weaning from TPN. Pancreaticobiliary secretions and intestinal hormones are as factors for process of intestinal adaptation. [2] Many hormones have been studied in SBS such as Growth hormone (GH), insulin-like growth factor family (IGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF). [3] The IGF family consist of three peptides; insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). [4] Some studies suggested that insulin has positive effects on the intestinal structure and absorptive function. [5],[6],[7] It had been used whether oral or subcutaneous route in these studies. [1],[3]

We aimed to compare the intestinal adaptation effects of subcutaneous and oral administration of insulin in rats with short bowel syndrome and their differences.


   Materials and Methods Top


Thirty Wistar albino rats (200-250 g) were used in this study with the approval of the Animal Research and Ethic Committee of Ondokuz Mayis University. Rats were kept in room temperature and were allowed water and liquid diet ad libitum. All rats underwent laparotomy and 75% bowel resection with re-anastomosis.

They randomly assigned equally to three groups; first group (n = 10) (SBS) was the control group. They had no further intervention. The second group (n = 10) was the subcutaneous insulin group with SBS (SI/SBS). In this group, insulin was applied subcutaneously at a dose of 1 U/kg, twice-daily, from day 3 to day 14. The third group (n = 10) was oral insulin group with SBS (OI/SBS). In this group, insulin was put in drinking water (1 U/ml) from day 3 to 14.

Laparotomy was performed in all rats with midline incision via intramuscular ketamin (3 mg/kg). A 75% bowel resection between 5 cm distal to ligament of Treitz and 10 cm proximal to the ileocecal valve was performed for SBS. After bowel resection, intestinal continuity was restored by end-to-end anastomosis. The rats were killed on day 15 by intramuscular ketamin injection. All rats' bowel weight was measured (g/cm/100 g), and specimens from both sides of anastomosis were obtained for histopathological and biochemical evaluation.

The intestine specimens were fixed in 10% buffered neutral formalin solution for 24 hours. Formalin-fixed tissues were embedded in paraffin and sectioned at a thickness of 4-6 μm. The sections were stained with haematoxylin-eosin and examined under light microscope. In each section, at least three villous height and crypt length parallel to each other were measured with samba analysis system morfometry module.

Each tissue was homogenized in liquid nitrogen and then sonicated for one minute 220 volt in 2 ml phosphate buffer (10 mM, pH 7,4). Homogenized tissues were stored until examination at -40°C. At the working day, frozen material was waited to melt at room temperature. After melting, homogenized tissues were centrifugated for 5 minutes at 3000 g, and separated supernatant were used for measurement.

Bender MedSystems ELISA kits (Human VEGF-C ELISA BMS297, Human GCSF BMS283 Vienna, Austria) were used for VEGF-C and GM-CSF measurement in the supernatant.

Principle of VEGF-C measurement; VEGF-C present in supernatant binds to antibodies adsorbed to the microwells. A biotin-conjugated policlonal VEGF-C antibody is added and binds to VEGF-C captured by receptors. Then, Streptavidin-HRP is added and binds to the biotin-conjugated VEGF-C. Substrate solution reactive with HRP is added to the wells. A coloured product is formed in proportion to the amount of VEGF-C present in the supernatant. The reaction is terminated by addition of acid, and absorbance is measured at 450 nm.

Principle of GM-CSF measurement; GM-CSF present in supernatant binds to antibodies adsorbede to the microwells. A HRP-conjugated monoclonal anti-GM-csf antibody is added and binds to GM-csf captured by the first antibody. Then, substrate solution reactive with HRP is added to the wells. A coloured product is formed in proportion to the amount of GM-CSF present in the supernatant. The reaction is terminated by addition of acid, and absorbance is measured at 450 nm.

The level of protein concentration in supernatant was measured by Lowry method. [8]

All results were presented as pg/mg protein.

Statistical analysis

For the statistical analysis, SPSS 13.0 was used. After normality analysis, ANOVA or Kruskal Wallis variance analysis was made. The groups were compared with t-test or Mann Whitney U tests, if the distribution was not normal.


   Results Top


The main results were shown on the [Table 1] and [Table 2]. Intestinal weight was significantly more in (SI/SBS) group and (OI/SBS) than in the control group (P < 0.05). Villus length and crypt depth values in both proximal and distal parts were higher in (SI/SBS) group than in other two groups, but there was no statistical difference (P > 0.05). There was also no difference of villus/crypt ratio between groups (P > 0.05). Typical histologic images of the groups are shown in [Figure 1]a-c.
Figure 1: (a) Histologic image of Control group (Hematoxylin eosin staining, x200). (b) Histologic image of oral insulin group (Hematoxylin eosin staining, x200). (c) Histologic image of subcutaneous insulin group (Hematoxylin eosin staining, x200)

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Table 1: Measurments of villous length and crypt depth of groups

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Table 2: Intestinal weight of groups, VEGF and GM-CSF values

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Biochemically, vascular endothelial growth factor (VEGF) value was not statistically significant between the groups (P > 0.05). Granulocyte-macrophage colony-stimulating factor (GMCSF) was statistically more in the control group than in the subcutaneous groups (P < 0.05). GMCSF was also statistically more in the oral group than in the subcutaneous group (P < 0.05).


   Discussion Top


Short bowel syndrome (SBS) is massive small bowel resection resulting in an inability to absorb adequate nutrients. The management goal of SBS is to provide adequate nutrition for patient and adaptation of intestine. Affected patients mostly require long-term parenteral nutrition (TPN). Following the SBS, gastrointestinal system undergoes a process of morphological and functional adaptation in terms of intestinal length, diameter and epithelial hyperplasia. [9],[10] In SBS, the adaptive capacity of residual bowel determines the duration of TPN treatment and recovery rate. [11],[12]

Many growth and trophic agents have been mentioned playing a role in this adaptation process. [13],[14],[15] One of the growth factors is insulin-like growth factor (IGF) family such as insulin, IGF-I and IGFII. Several studies have shown the positive role of insulin in the intestinal trophic effect. [4],[5],[16] In rat models, IGF-I administration improves weight gain and growth of small bowel mucosa after small bowel resection. [17] It has been also shown that IGF-I has positive effects on mucosal DNA to promote cell proliferation and crypt depth of jejunal mucosa. [18],[19]

In the present study, we demonstrated that insulin has positive effect on hyperplasia of intestine in SBS. Although we could not be able to show statistical difference between groups according to villus length, crypt depth or rate of those, particularly subcutaneous insulin, is beneficial in this aspect. Our results were similar to the Sukhotnik and Ben Lulu. [4],[5]

Granulocyte-macrophage colony-stimulating factor is a cytokine that promotes myeloid cell development and maturation. [20] GM-CSF is effective in treatment of inflammatory bowel disease and promote colonic mucosal restore. [21],[22] In the current study, GM-CSF level was lowest in subcutaneous insulin group and was statistically significant. We suggest that GM-CSF depletion indicates the intestinal mucosal proliferation.

Vascular epidermal growth factor (VEGF) is an endogenous peptide and stimulates the intestinal mucosal restore. It may augment the adaptative process and diminish the apoptosis following the small bowel resection. [15],[23] We did not demonstrate any significant differences of VEGF level between groups, but the lowest rate was in the subcutaneous insulin. This result led us to think subcutaneous insulin application has superiority on other groups in terms of intestinal adaptation effect.

Lack of further groups with insulin administration and no SBS are the major limitations of our study.

As conclusion, administration of insulin either oral or subcutaneous has some beneficial effects in rats with SBS. Subcutaneous insulin seems to be more effective than the oral route. The findings of the current study promise improvements of SBS in clinical setting.

 
   References Top

1.Wales PW, de Silva N, Kim JH, Lecce L, Sandhu A, Moore AM. Neonatal short bowel syndrome: A cohort study. J Pediatr Surg 2005;40:755-62.  Back to cited text no. 1
[PUBMED]    
2.McMellen ME, Wakeman D, Longshore SW, McDuffie LA, Warner BW. Growth factors: Possible roles for clinical management of the short bowel syndrome. Semin Pediatr Surg 2010;19:35-43.  Back to cited text no. 2
[PUBMED]    
3.Nucci AM, Finegold DN, Yaworski JA, Kowalski L, Barksdale E. Results of growth trophic therapy in children with short bowel syndrome. J Pediatr Surg 2004;39:335-9.  Back to cited text no. 3
    
4.Sukhotnik I, Mogilner J, Shamir R, Shehadeh N, Bejar J, Hirsh M, et al. Effect of subcutaneous insulin on intestinal adaptation in a rat model of short bowel syndrome. Pediatr Surg Int 2005;21:132-7.  Back to cited text no. 4
[PUBMED]    
5.Ben Lulu S, Coran AG, Mogilner JG, Shaoul R, Shamir R, Shehadeh N,et al. Oral insulin stimulates intestinal epithelial cell turnover in correlation with insulin-receptor expression along the villus-crypt axis in a rat model of short bowel syndrome. Pediatr Surg Int 2010;26:37-44.  Back to cited text no. 5
    
6.Lemmey AB, Martin AA, Read LC, Tomas FM, Owens PC, Ballard FJ.IGF-I and truncated analogue des-(1-3) IGF-I enhance growth in rats after gut resection. Am J Physiol 1991;260(2 Pt 1):E213-9.  Back to cited text no. 6
    
7.Lund PK. Moleculer basis of intestinal adaptation: The role of the insulin-like growth factor system. Ann N Y Acad Sci 1998;859:18-36.  Back to cited text no. 7
[PUBMED]    
8.Lowry OH, Rosenbrough NJ, Farr AL, Randal RJ. Protein measurement with the folinphenol reagent. J Biol Chem 1951;193:265-75.  Back to cited text no. 8
    
9.Donohoe CL, Reynolds JV. Short bowel syndrome. Surgeon 2010;8:270-9.  Back to cited text no. 9
[PUBMED]    
10.Soden JS. Clinical assessment of the child with intestinal failure. Semin Pediatr Surg 2010;19:10-9.  Back to cited text no. 10
[PUBMED]    
11.Jenkins AP, Thomson RP. Mechanisms of small intestinal adaptation. Dig Dis 1994;12:15-27.  Back to cited text no. 11
    
12.Washizawa N, Gu LH, Gu L, Openo KP, Jones DP, Ziegler TR. Comparative effects of glucagon-like peptide-2 (GLP-2), growth hormone (GH), and keratinocyte growth factor (KGF) on markers of gut adaptation after massive small bowel resection in rats. JPEN J Parenter Enteral Nutr 2004;28:399-409.  Back to cited text no. 12
[PUBMED]    
13.Lentze MJ. Intestinal adaptation in short bowel syndrome. Eur J Pediatr 1989;148:294-9.  Back to cited text no. 13
[PUBMED]    
14.Gu Y, Wu ZH. The anabolic effects of recombinant human growth hormone and glutamine on parenterally fed, short bowel rats. World J Gastroenterol 2002;8:752-7.  Back to cited text no. 14
[PUBMED]    
15.Stern LE, Erwin CR, O'Brien DP, Huang F, Warner BW. Epidermal growth factor is critical for intestinal adaptation following small bowel resection. Microsc Res Tech 2000;51:138-48.  Back to cited text no. 15
    
16.Ohnedo K, Ulshen MH, Fuller CR, D'Ercole AJ, Lund PK. Enhanced growth of small bowel in transgenic mice expressing human insulin-like growth factor I. Gastroenterology 1997;112:444-54.  Back to cited text no. 16
    
17.Gillingham MB, Dahly EM, Murali SG, Ney DM. IGF-I treatment facilitates transition from parenteral to enteral nutrition in rats with short bowel syndrome. Am J Physiol Regul Integr Comp Physiol 2003;284:R363-71.   Back to cited text no. 17
[PUBMED]    
18.Peterson CA, Carey HV, Hinton PL, Lo HC, Ney DM. GH elevates serum IGF-I levels but does not alter mucosal atrophy in parenterally fed rats. Am J Physiol 1997;272(5 Pt 1):G1100-8.  Back to cited text no. 18
    
19.Ozen S, Akisu M, Baka M, Yalaz M, Sozmen EY, Berdeli A, et al. Insulin-like growth factor attenuates apoptosis and mucosal damage in hypoxia/reoxygenation- induced intestinal injury. Biol Neonate 2005;87:91-6.  Back to cited text no. 19
[PUBMED]    
20.Egea L, Hirata Y, Kagnoff MF. GM-CSF: A role in immune and inflammatory reactions in the intestine. Expert Rev Gastroenterol Hepatol 2010;4:723-31.  Back to cited text no. 20
[PUBMED]    
21.Krishnan K, Arnone B, Buchman A. Intestinal growth factors: Potential use of in the treatment of inflammatory bowel disease and their role in mucosal healing. Inflamm Bowel Dis 2011;17:410-22.  Back to cited text no. 21
[PUBMED]    
22.Bernasconi E, Favre L, Maillard MH, Bachmann D, Pythoud C, Bouzourene H, et al. Granulocyte-macrophage colony stimulating factor elicits bone marrow derived cells that promote efficient colonic mucosal healing. Inflamm Bowel Dis 2010;16:428-41.  Back to cited text no. 22
[PUBMED]    
23.Parvadia JK, Keswani SG, Vaikunth S, Maldonado AR, Marwan A,Stehr W, et al. Role of VEGF in small bowel adaptation after resection: The adaptive response is angiogenesis dependent. Am J Physiol Gastrointest Liver Physiol 2007;293:G591-8.  Back to cited text no. 23
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Correspondence Address:
Burak Tander
Department of Pediatric Surgery, Ondokuz Mayis University, Samsun
Turkey
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Source of Support: None, Conflict of Interest: None of the authors has any confl ict of interest to disclosure.


DOI: 10.4103/0189-6725.115030

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