Glucagon-like peptide-2 exhibits protective effect on hepatic ischemia-reperfusion injury in rats

Abstract

Glucagon-like peptide-2 (GLP-2) has potent anti-inflammatory effects and protects against experimental ischemia/reperfusion (I/R) injury in pulmonary, intestinal, and myocardial tissue. However, its protective abilities against I/R injury in the liver are unknown. We investigated the potential role of GLP-2 pretreatment on hepatic I/R injury in rats. A total of 24 rats were randomly divided into three groups (n = 8). The first group was the control group; the second group was the vehicle-treated hepatic ischemia/reperfusion (HIR, vehicle saline-treated) group; and the third group was the GLP-2 pretreated I/R (GLP2-IR) group. Each rat in the third group was intraperitoneally administered 5 µg GLP-2 for 5 d before the procedure. A portal triad was created to induce ischemia with a vascular atraumatic clamp. After 40 min, the clamp was released to initiate hepatic reperfusion for 6 h. Blood samples and tissue specimens from the liver were obtained. Alanine aminotransferase, aspartate aminotransferase, and total bilirubin levels significantly increased in the saline-treated HIR group (P<0.001), whereas GLP-2 pretreatment significantly decreased their levels (P<0.01). Our data suggested that GLP-2 pretreatment may have a protective effect on liver I/R injury. However, dose-response studies are necessary to determine the most effective dose.

Keywords

ischemia/reperfusion / liver / glucagon-like peptide-2 / alanine aminotransferase

Introduction

Ischemia/reperfusion (I/R) injury is caused by the return of blood supply after long-term ischemia [ 1]. It is one of the main causes of postoperative liver failure and primary graft non-function, leading to increased rates of mortality in liver surgery and transplantation [ 2]. Sepsis, respiratory failure, congestive cardiac failure, trauma, and hemorrhage can also cause I/R injury in liver as a result of hypoxia and hypovolemia [ 1, 3, 4]. The critical period of ischemia varies among organs; this period is approximately 15-20 min for the liver [ 5]. Tissue damage can be reversible or irreversible depending on the blood supply of the organ or tissue after ischemia. Reperfusion is essential to prevent irreversible tissue damage. However, reperfusion can lead to more damage compared with ischemia [ 6].

The pathology of liver I/R injury involves various mechanisms that can be summarized as follows: activation of Kupffer cells, production of reactive oxygen species, increased secretion of cytokines and chemokines, vasoconstriction, impairment of balance between nitric oxide and endothelia, migration of neutrophils, changes in mitochondrial permeability, influx of Ca²⁺ into cells, and changes in pH of milieu [ 7- 9]. Cellular and humoral factors both play roles in I/R injury. Kupffer cells are activated and produce reactive free radicals and proinflammatory cytokines. The major component of the proinflammatory cascade is TNF-α, and the other important cytokines are IFN-γ, IL-1β, IL-6, IL-10, IL-12, IL-13, IL-18, and VEGF [ 1].

GLP-2 is a single-chain polypeptide produced by enteroendocrine L cells. Enteroendocrine L cells are primarily found in the terminal ileum and the colon, but can also be found throughout the gastrointestinal (GI) tract. External GLP-2 on the GI tract stimulates blood flow [ 10- 13] and increases barrier function in the intestines [ 14, 15]. Its anti-inflammatory effects are also reported in literature [ 16, 17].

The actions of GLP-2 are manifested by a specific G protein-coupled receptor (GLP-2R) largely expressed in the GI tract [ 18]. GLP-2R is mainly found in the jejunum, but is also in the ileum, colon, and stomach. Although GLP-2R expression is absent in crypt epithelial cells or enterocytes, its effects on intestinal growth have been suggested to proceed via an indirect mechanism. Recently, GLP-2 has been demonstrated to have anti-inflammatory effects on the intestinal mucosa. However, these effects are suggested to occur via pathways besides trophic effects [ 16]. The presence of GLP-2Rs in the hepatic system has not been established, but an uptake of radiolabeled GLP-2 in the liver has been shown in a previous study [ 19].

The reduced inflammation observed with GLP-2 treatment has been associated with decrease in expressions of TNF-α, IFN-γ, and IL-1β in the local inflammatory milieu [ 16].

Many agents have been used in liver I/R injury, such as steroids, pentoxifylline, α-tocopherol, and allopurinol [ 20- 24]. Glucagon-like peptide-1, a co-released hormone, has been shown to have a protective effect on liver I/R injury [ 25]. However, the effects of GLP-2 on liver I/R injury have not been studied to the best of our knowledge. Based on the abovementioned effects of GLP-2, this peptide possibly exerts protective effects under I/R conditions. Therefore, we aimed to investigate whether GLP-2 has protective effects on hepatic I/R injury in a hepatic I/R rat model.

Materials and methods

Animals

A total of 24 Sprague-Dawley rats, weighing 240-260 g, were housed under standard animal care conditions with free access to food and water and kept under controlled environmental conditions with a 12 h light and dark cycle. Animal care and all procedures were approved by the Animal Care Committee (13.02.2013/2013-02-01) of Çanakkale Onsekiz Mart University.

Hepatic IR injury model and experimental protocol

The rats were randomly divided into three groups (n = 8), namely, the control group, the hepatic I/R (HIR, vehicle saline-treated) group, and the GLP-2 pretreated I/R (GLP2-IR) group. GLP-2 (Sigma-Aldrich Corp., St. Louis, MO, USA) was intraperitoneally administered at 5 µg/rat once a day for 5 d before I/R.

Before all the surgical procedures were performed, 50 mg/kg ketamine hydrochloride (Ketalar®, Parke–Davis, Eczacibasi, Istanbul, Turkey) and 10 mg/kg xylazine (Rompun®, Bayer AG, Leverkusen, Germany) were intramuscularly administered. A midline incision was made, after which ischemia was induced by clamping the portal triad structures with a vascular atraumatic clamp. After 40 min, the clamp was released to initiate hepatic reperfusion for 6 h. Animals were sacrificed at the end of the reperfusion period, and blood was taken from the heart. Tissue samples were taken from the liver for histopathological analysis. The same protocol without vascular occlusion was performed in the control rats.

Liver enzyme levels

Serum was collected 6 h post-reperfusion and kept on ice until processed. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, total bilirubin, alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT) were determined in serum. Routine biochemical measurements were performed on the same day using enzymatic and colorimetric methods on the Cobas® c 501 module of the Cobas® 6000 analyzer (Roche Diagnostics International, Ltd., Rotkreuz, Switzerland).

Histopathological evaluation

Liver specimens from all groups were fixed in 10% formalin, embedded in paraffin blocks, and stained with hematoxylin-eosin. Pathological findings were assessed by a researcher blinded to the group allocations. An ordinal scale was used to evaluate severity of hepatic injury: grade 0 = minimal to no evidence of injury; grade 1 = mild injury with cytoplasmic vacuolation and focal nuclear pyknosis; grade 2 = moderate to severe injury with enlarged nuclear pyknosis, cytoplasmic hypereosinophilia, and loss of intercellular borders; grade 3 = severe necrosis with disintegration of hepatic cords, hemorrhage, and neutrophil infiltration [ 26].

Statistical analysis

The Statistical Package for the Social Sciences 16.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All values were given as mean ± standard deviation. Kruskal-Wallis test was used for variance analyses. Mann-Whitney U test with Bonferroni correction was used for dual comparisons between groups. Statistical significance was accepted at P < 0.05.

Results

All animals survived throughout the experimental procedures.

Effect of GLP-2 on liver enzymes in blood

Serum levels of biochemical parameters are shown in Table 1.

The ALT levels in HIR, vehicle saline-treated group significantly increased compared with the control group (P < 0.001), whereas GLP-2 pretreatment significantly lowered the ALT levels (P < 0.001).

The AST levels were significantly higher in HIR, vehicle saline-treated group compared with the control group (P < 0.001), whereas GLP-2 pretreatment significantly lowered the AST levels (P < 0.001).

The GGT levels significantly increased in HIR, vehicle saline-treated group compared with the control group (P < 0.01). GGT levels decreased in the GLP-2 pretreated group, but the decrease was not statistically significant (P > 0.05).

The ALP levels significantly increased in HIR, vehicle saline-treated group (P < 0.01). In the GLP-2 pretreated group, the ALP levels decreased but this decline was not statistically significant (P > 0.05).

Total bilirubin levels significantly increased in HIR, vehicle saline-treated group (P < 0.001) but significantly decreased in GLP-2 pre-treated group (P < 0.001) as ALT and AST levels.

No differences in total protein levels were observed among the groups (P > 0.05).

Effect of GLP-2 on liver histology after I/R injury

No pathological changes in the liver tissue of the control group were observed. Focal necrosis and leukocyte infiltration were observed in liver specimens of saline-treated I/R group. GLP-2 treatment did not change these pathological scores (Fig. 1).

The liver histopathological score was 0.062 ± 0.018 in the control group, 0.75 ± 0.25 in vehicle-treated I/R group, and 0.75 ± 0.16 in the GLP2-IR group. The histopathological scores were significantly higher in the I/R groups than in the control group (P < 0.05 in all cases) (Fig. 2).

Discussion

In the present study, GLP-2 pretreated rats had significantly lower serum levels of I/R-dependent liver enzymes. The studied polypeptide, GLP-2, has been shown to have potent anti-inflammatory effects [ 16, 17]. GLP-2 has been shown to be protective in experimental ischemia/reperfusion (I/R) injuries in pulmonary, intestinal, and myocardial tissue, but whether it is protective for other organs is unknown. In addition, GLP-2R is found in other sites of the body aside from the GI system, such as in the hypothalamus, suggesting that GLP-2 might be protective against ischemia in other tissue [ 18]. Thus, we examined the effect of GLP-2 in hepatic I/R injury in rats. GLP-2 pretreated rats had significantly lower serum AST, ALT, and total bilirubin levels than vehicle saline-treated rats.

Ischemic hypoxia is caused by the retention of metabolites in the bloodstream and the increase in ATP consumption. These lead to lactic acid production and decrease in venous oxygen saturation [ 27, 28]. TNF-α is the most important cytokine in hepatic I/R injury [ 16]. High levels of TNF-α have been shown to be associated with cell death [ 29]. Apoptosis plays a central role in liver I/R injury [ 30, 31]. We assumed that GLP-2 can decrease apoptosis in liver cells because of its suppressive effect on TNF-α. We were not able to determine TNF-α levels and apoptosis.

Another important source of free oxygen radicals is arachidonic acid metabolism. Various free radicals and cyclic endoperoxides arise from the separation of arachidonic acid from cell membrane phospholipids and its subsequent enzymatic oxidation. Non-steroidal anti-inflammatory agents have been shown to inhibit the formation of cyclooxygenase products in arachidonic acid metabolism [ 32]. We aimed to investigate the anti-inflammatory effect of GLP-2 on ischemic injury via arachidonic acid metabolism. However, no difference in histopathology was detected among the experimental groups.

Transaminases catalyze the transfer of amino acids from aspartate and alanine to ketoglutaric acid during oxaloacetate and pyruvate production in gluconeogenesis in the liver. These enzymes are the best markers of hepatocellular injury. ALT is an intracytoplasmic enzyme that is a specific marker for liver injury. By contrast, AST is found in the cytoplasm and mitochondria, as well as in the tissue other than the liver, such as the myocardium, skeletal muscle, erythrocytes, kidney, pancreas, lung, and brain. Therefore, its specificity for the liver is lower than that of ALT. In the present study, both ALT and AST levels significantly decreased with GLP-2 treatment. As shown in previous studies, the mitogenic effect of GLP-2 could be responsible for this process.

ALP is another enzyme found in lots of tissue. In the liver, it is present on the sinusoidal and canalicular membranes as well as in the cytoplasm. Its serum levels increase when the bile flow is blocked. Although ischemia increased its levels in our study, GLP-2 treatment had no significant effect on ALP levels.

GGT is a microsomal enzyme that increases in both cholestatic and liver diseases. In the present study, its levels increased after ischemia and decreased with GLP-2 treatment. However, this decline was not statistically significant.

Bilirubin levels can increase both in hepatocellular injury and in cholestatic liver diseases. In addition, extrahepatic bile flow obstructions increase bilirubin levels. We found that increased total bilirubin levels after ischemia decreased significantly with GLP-2 treatment. Thus, GLP-2 could have a vasodilator effect on canalicular smooth muscles.

In recent years, various agents were experimentally used to protect I/R injury. Tsung et al. [ 33] found that ethyl pyruvate inhibits hepatic necrosis and apoptosis in liver I/R injury. Ethyl pyruvate was intravenously administered at a dose of 100 mg/kg 1 h prior to ischemia, immediately before ischemia, and before reperfusion. AST and ALT levels decreased substantially and histopathology remarkably improved. Human recombinant erythropoietin was shown to reduce the levels of transaminases markedly in liver I/R injury in rats [ 34]. Erdoğan et al. [ 35] investigated the effect of verapamil in experimental liver I/R injury, and found that intraportal verapamil significantly decreased the transaminase levels in liver I/R injury. Hiranuma et al. [ 36] carried out 90 min liver ischemia and 24 h reperfusion in rats. Edaravone was given 5 min before reperfusion. Both ALT and AST levels and histopathological scores significantly improved. Yıldırım et al. [ 24] used allopurinol and pentoxifylline, showing that the transaminases decrease significantly in these groups than in the control group. In our study, ALT, AST, and total bilirubin levels were significantly lower in the GLP-2 pretreated group.

Taylor-Edwards et al. [ 37] showed that GLP-2 treatment increased hepatic blood flow in calves. Guan et al. [38] showed an increase in blood flow of liver in TPN-fed piglets by GLP-2. In the same study, GLP-2 decreased activity of nitric oxide synthetase and increased hepatic blood flow in a nitric oxide-dependent manner. The beneficial effects of GLP-2 are related to increased blood flow.

However, based on our pathological results, GLP-2 did not significantly decrease hepatocellular injury.

Limitations of the study are that we could not study other oxidative stress markers, such as TNF-α, glutathione, or myeloperoxidase.

In conclusion, GLP-2 could prevent ischemic liver damage. Our results suggested that GLP-2 treatment may be used in the hepatic I/R injuries. However, the dose and treatment period need to be investigated comprehensively in future works to support the role of GLP-2 in protection against hepatic I/R injury and to verify its potential clinical use.

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