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Immunohistochemical, apoptotic and biochemical changes by dipeptidyl peptidase-4 inhibitor-sitagliptin in type-2 diabetic rats
Pharmacological Reports 67 (2015) 846–853
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Immunohistochemical, apoptotic and biochemical changes by dipeptidyl peptidase-4 inhibitor-sitagliptin in type-2 diabetic rats Sezin Karabulut a, Zeynep Mine Coskun b, Sema Bolkent a,* a b
Department of Medical Biology, Faculty of Cerrahpasa Medicine, Istanbul University, Istanbul, Turkey Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Istanbul Bilim University, Istanbul, Turkey
A R T I C L E I N F O
Article history: Received 18 September 2014 Received in revised form 26 November 2014 Accepted 19 January 2015 Available online 1 February 2015 Keywords: Apoptosis Rat Sitagliptin Type-2 diabetes Pancreas
A B S T R A C T
Background: Diabetes is a major public health problem that is rapidly increasing in prevalence. In this study, the effects of sitagliptin, a dipeptidyl peptidase-4 inhibitor, were examined on newborn diabetic rat model. Methods: Wistar albino newborn rats were divided into control (Ctrl), sitagliptin (Sit), diabetic and diabetic + Sit groups. On the second day after the birth, 100 mg/kg streptozotocin (STZ) was administered intraperitoneally in a single dose to induce type-2 diabetes in rats. The Sit and diabetic + Sit groups were administered sitagliptin (1.5 mg/kg subcutaneous) as of the day 5 for 15 days. The pancreas sections were stained with insulin (INS), glucagon (GLU), somatostatin (SS), glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-1 receptor (GLP-1R) antibodies by the streptavidin–biotin peroxidase technique. The TUNEL method for apoptosis and biochemical analysis were performed in the pancreas and serum, respectively. Results: Body weight and blood glucose levels showed significant differences among all groups on days 11 and 20. In diabetic rats following treatment with sitagliptin, the area percentage of INS immunopositive cells increased while the area percentage of SS immunopositive cells decreased, insignificantly. A significant increase was observed on the area percentage of GLU, GLP-1 and GLP-1R immunopositive cells in the diabetic + Sit group when compared to the diabetic group. The area percentage of apoptotic cells was the same among all groups. While serum glutathione and malondialdehyde levels demonstrated insignificant alterations, the catalase and superoxide dismutase activity significantly changed among four groups. Conclusion: According to our findings, sitagliptin may be a useful therapeutic agent to a certain extent of type-2 diabetic condition. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction Diabetes mellitus is one of the most common metabolic diseases in the world that is characterized by increased blood glucose level during fasting or oral glucose tolerance test [1,2]. Type-2 diabetes is identified by means of impairments in the b-cell function and insulin (INS) secretion [1,3]. Streptozotocin (STZ) is widely preferred in investigations as part of studies of the type-2 diabetic model [4]. In animal models, STZ is administered to newborn rats to induce type-2 diabetes [5].
* Corresponding author. E-mail addresses: [email protected], [email protected] (S. Bolkent).
Incretin hormones are important to understand the mechanism of glucose homeostasis. Glucagon-like peptide-1 (GLP-1), one of the most important of incretin hormones, increases insulin secretion after food ingestion [6]. It is suggested that GLP-1 receptor (GLP-1R) is expressed on a and b cells of pancreas, gastrointestinal system, kidney, lung, heart and brain [7,8]. It has been reported that GLP-1 provides b cell regeneration, differentiation and prevents apoptosis via the activation of GLP-1R [9,10]. GLP-1 hormones are degraded by the dipeptidyl peptidase-4 (DPP-4) enzyme [11]. DPP-4 is expressed in pancreas, brain, lung, kidney, liver, intestine, adrenal gland and lymphocytes [12]. Long-term studies suggest that DPP-4 inhibitors decreased b cell death, increase its neogenesis and function in diabetic animals [13,14]. Sitagliptin, a novel DPP-4 inhibitor, is a therapeutic agent for type-2 diabetes that is administered either as a monotherapy or
http://dx.doi.org/10.1016/j.pharep.2015.01.010 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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combination therapy [15,16]. Furthermore, the agent provides improvements in glycemic control [17]. DPP-4 inhibitors-related studies on humans have found a decrease in HbA1c levels [18]. It has been reported that DPP-4 inhibitors do not affect body weight [19]. Moreover, it is suggested that sitagliptin may have a role as a treatment agent for pre-diabetes [20]. In our study, we aimed to explore the biochemical alterations in serum, INS, glucagon (GLU), somatostatin (SS), GLP-1 and GLP-1R expressions and programmed cell death (apoptosis) in the pancreas of healthy and type-2 diabetic rats following sitagliptin administration.
sections were prepared by means of substituting antibodies with phosphate buffer saline (PBS). The rat stomach sections were used as SS positive controls and non-experimental rat pancreas sections were used as INS, GLU, GLP-1 and GLP-1R positive controls because of their high expressions. The photographs of the Langerhans islets in pancreas were taken and the size of islets calculated using a Nikon Eclipse 80i light microscope equipped with a digital camera using NIS-Elements-D 3.1 microscope imaging software program.
Materials and methods
The apoptosis was determined by the terminal deoxynucleotidyl transferase (Tdt) mediated dUTP (TUNEL) method (APOPTAG1 kit; Millipore S7101) according to the manufacturer’s instructions. The reaction was observed using an AEC substrate kit. The negative control sections were prepared by substituting Tdt enzyme with distilled water. The rat breast tissue sections (S7115) were used as positive controls for the detection of programmed cell death.
Animals and tissue preparation
Apoptosis
All animal experimental procedures were approved by the Istanbul University Local Ethics Committee on Animal Research. When the newborn Wistar albino rats were 48-h-old following birth, they were included in the experimental groups. There were four groups of newborn rats. Group I (n = 8): the control group (Ctrl) consisted of newborn rats that were given physiological saline for a period of 18 days, intraperitoneally (ip). Group II (n = 8): the sitagliptin group (Sit) was treated with 1.5 mg/kg/day sitagliptin (Januvia, Merck) dissolved in physiological saline from the fifth day for a period of 15 days, subcutaneously (sc). Group III (n = 8): for streptozotocin (STZ)-induced diabetic rats (diabetic), 100 mg/kg STZ (Sigma–Aldrich, S0130) was dissolved in physiological saline and given as a single intraperitoneal dose on the second day following birth [21,22]. The rats whose blood glucose levels were 200 mg/dl or more on the second day following STZ injection were considered as diabetic. Group IV (n = 8): the diabetic group which was given sitagliptin (diabetic + Sit) on the fifth day following birth for a period of 15 days. The blood glucose levels of animals in all groups were measured with a glucometer (Accu-check, Roche Diagnostics GmbH) using the blood samples collected from the tail vein on days 2, 4, 11 and 20. The body weights of the animals were also measured at the same time intervals. On the 20th day, the animals were left to fast for 1 h; blood and pancreatic tissue samples were collected under ketamin-HCl (50 mg/kg) anesthesia. The tissue samples were fixed in 10% neutral buffered formalin for 24 h at +4 8C and then embedded in paraffin using routine light microscopy processing methods.
The blood samples were then centrifuged at 3000 rpm for 10 min to separate the serum. The biochemical parameters were determined on serum samples were frozen at 80 8C. Serum glutathione (GSH), malondialdehyde (MDA) levels, catalase (CAT) and superoxide dismutase (SOD) activities were measured. GSH levels were determined according to Beutler’s method using metaphosphoric acid for protein precipitation and 5,50 -dithiobis (2-nitro benzoic acid (Sigma–Aldrich) for the color development at 412 nm [24]. The MDA levels were estimated by using the Ledwozyw’s methods [25]. The serum samples were mixed thoroughly with a solution of trichloroacetic acid (TCA) (Sigma– Aldrich) (30%), TBA (Merck) (0.75%) and 5 M hydrochloric acid. The samples were measured at 535 nm. The CAT activity was assayed by the methods of Aebi. The reaction mixture was made up of the sample, 50 mM phosphate buffer pH 7.0 and 10 mM H2O2. The reduction rate of H2O2 was observed to be 240 nm at the room temperature for 60 s [26]. The SOD activity was determined by the method developed by Beauchamp and Fridovich that involves the inhibition of nitroblue tetrazolium (NBT) (Sigma–Aldrich, Louis) [27]. The protein content in the serum was estimated by the method of Lowry [28].
The study of Langerhans islet size and immunohistochemical staining
Statistical analysis
The sections of pancreases were stained by using hematoxylin and eosin. All of the Langerhans islets in pancreas were classified as small, medium and large according to their size after calculating the areas. Large islets were >10,000 m2, medium islets were >5000–10,000 m2, and small islets were <5000 mm2 [23]. For immunohistochemical staining, the sections were incubated with mouse monoclonal INS (dilution 1:1000; Sigma–Aldrich, I2018), mouse monoclonal GLU (dilution 1:2000; Sigma–Aldrich, G2654) and rabbit polyclonal SS (dilution 1:2500; Chemicon, AB1976), mouse monoclonal GLP-1 (dilution 1:500; Santa Cruz, sc57166) and rabbit polyclonal GLP-1R (dilution 1:50; Novus, NLS1205) antibodies. They were stained with the streptavidin– biotin-peroxidase method using histostain-plus bulk kit (Zymed 85-9043). The detection procedures were carried out as described by the manufacturer. The enzyme activity was developed using 3-amino-9-ethyl-carbazole (AEC) substrate kit (Zymed 00-2007) and the sections were then counterstained with Mayer’s hematoxylin. The staining intensity was scored from one to three as weak (+), medium (++) and strong (+++). The negative control
The INS, GLU, SS, GLP-1, GLP-1R immunopositive cells and apoptotic cells were counted in all islets of Langerhans for each section of rat pancreas. The area percentages of immunopositive and apoptotic cells in the islets were calculated by using the formula of (labeling area/total area) 100. The values obtained were evaluated statistically. SPSS 21.0 software was used for the statistical analysis. The results were expressed as means SEM. The statistical evaluations were performed using Kruskal–Wallis test followed by Mann– Whitney U test. A p-value <0.05 was considered to be significant.
Biochemical assay
Results Body weight and blood glucose In Figs. 1 and 2, the changes in the comparable body weight and blood glucose levels for all experimental groups are presented. A significant difference in body weight and blood glucose level was
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Body weight (g)
40
a,c
30
b,c
a,c
a,c
20
C Sit STZ-diabetic STZ-diabetic + Sit
a
a
10
20
11 D
D
ay
ay
4 ay D
D
ay
2
0
Fig. 1. The changes of body weights (g) in experimental rats on days 2, 4, 11 and 20, n = 8 in each group. Data are expressed as means SEM. ap < 0.001 vs. control (Ctrl) group, bp < 0.01 vs. Ctrl group, cp < 0.001 vs. sitagliptin (Sit) group. Versus: vs.
Blood Glucose (mg/dl)
400
C Sit STZ-diabetic STZ-diabetic + Sit
300 200
b,c
a,b b,c
b,c
100
immunopositive islet area percentage increased significantly in the diabetic group as compared to Ctrl group (p < 0.05). The GLU area percentage of the islet significantly increased in the diabetic + Sit group as compared to the diabetic group (p < 0.01) (Fig. 3). There was no significant difference among four groups with respect to the area percentage of SS immunopositive cells (Fig. 4). The immunopositive cells area percentage of GLP-1 in the islets was almost the same in the diabetic group as compared to the Ctrl group. The area percent of GLP-1 in the islet significantly increased in the diabetic + Sit group when compared to diabetic (p < 0.05) (Fig. 5). The GLP-1 immunopositive islet area significantly decreased in the Sit and diabetic groups as compared to Ctrl group, respectively (p < 0.001, p < 0.001). The GLP-1R percentage in the islet area was significantly increased in the diabetic + Sit group when compared to diabetic (p < 0.001). The GLP-1 peptide was located in islet periphery, while the GLP-1R was located both center and periphery of islet (Fig. 5). With respect to reaction intensity, the staining intensity of insulin immunopositive cells in diabetic + Sit group (++) was weaker than the other groups (+++). However, the staining intensity of glucagon (++), somatostatin (++), glucagon-like peptide-1 (+) and glucagon-like peptide-1 receptor (+) immunopositive cells and apoptotic cells (+++) did not vary between diabetic and diabetic + Sit groups. The programmed cell death by means of TUNEL method
20 D
ay
11 D
ay
4 ay D
D
ay
2
0
Fig. 2. Effects of sitagliptin on blood glucose (mg/dl) levels in rats on days 2, 4, 11 and 20, n = 8 in each group. Data are expressed as means SEM. ap < 0.01 vs. Ctrl group, bp < 0.001 vs. sitagliptin (Sit) group, cp < 0.001 vs. the Ctrl group. Versus: vs.
determined among all groups, respectively (pANOVA < 0.001, pANOVA < 0.001). There was a significant increase in body weights of diabetic group as compared to Ctrl group on days 4, 11 and 20, respectively (p < 0.001, p < 0.01, p < 0.001). However, it was observed that the body weight of the diabetic + Sit group decreased insignificantly when compared to diabetic rats on days 11 and 20. There was a significant increase in the blood glucose levels of diabetic rats as compared to Ctrl rats on days 4, 11 and 20, respectively (p < 0.001, p < 0.01, p < 0.001). It was determined that there were no changes in the blood glucose levels of diabetic + Sit group as compared to diabetic group on days 11 and 20. The results of Langerhans islet size measurement and immunohistochemistry The pancreas sections from Ctrl, diabetic and diabetic + Sit rats are shown in Figs. 3–5, respectively. It was seen that animals in the Ctrl group had the greatest area percent of large size islets as compared to the other groups. Diabetic + Sit rats had the highest percentage of small size islets when compared to the other groups. It was observed that diabetic + Sit group had more small size islets when compared to diabetic group (Table 1). The area percentage of INS, GLU, SS, GLP-1 and GLP-1R immunopositive cells for each group is presented in Table 2, respectively (pANOVA < 0.001, pANOVA < 0.001, pANOVA > 0.05, pANOVA < 0.05, pANOVA < 0.001). The area percentage of INS immunopositive cells significantly decreased in the diabetic group as compared to Ctrl group (p < 0.001). The INS area percentage in the islet of Langerhans increased insignificantly in the diabetic + Sit group as compared to diabetic rats (Fig. 3). The GLU
The TUNEL method was applied to pancreas sections with stained nucleus were evaluated as apoptotic cells (data not shown). The apoptotic cells percentage did not demonstrate a significant difference among four groups. It was evaluated that the reaction intensity of apoptotic cells was the same in islets of rats (+++). Biochemical results The effect of sitagliptin on GSH, MDA levels and CAT, SOD activities in serum of diabetic and Ctrl rats are seen in Table 3. The GSH level showed an insignificant increase in diabetic rats treated with sitagliptin as compared to other diabetic rats. There were no significant differences among all groups in terms of MDA levels. The MDA level showed an insignificant decrease in the diabetic + sitagliptin group as compared to the diabetic group. The CAT and SOD activity significantly varied among all groups, respectively (pANOVA < 0.05, pANOVA < 0.001). The CAT activity was found to be insignificantly increased in diabetic rats as compared to the Ctrl group. On the contrary, SOD activity showed a significant decrease in the diabetic group when compared to the Ctrl group (p < 0.05). Administration of sitagliptin to diabetic rats insignificantly decreased CAT activity and increased the SOD activity as compared to diabetic rats. Discussion The studies show that a decrease has been observed in body weights of diabetic adult rats as compared with healthy rats [29,30]. Conversely, it is suggested that there are no differences in body weight between healthy and diabetic rats at the end of third week after birth [5]. It is reported that sitagliptin did not change the body weights of different species of diabetic animals [31,32]. By contrast with the previous studies, administration of sitagliptin regulated the body weight of diabetic rats in this study. There was an extreme increase in body weight of diabetic rats on days 11 and 20. The body weight in diabetic rats approached the body weight of normal rats on days 11 and 20 with administration of sitagliptin. This difference supports the view that sitagliptin can
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Fig. 3. Insulin (INS) and glucagon (GLU) immunopositive cells (arrow) are observed by means of immunohistochemistry in the islets of Langerhans, n = 8 in each group. Streptavidin–biotin-peroxidase technique, counterstain hematoxylin. Scale bar = 10 mm.
be effective on the regulation of body weight in type-2 diabetic rats. The blood glucose level of both diabetic newborn and adult rats has been shown to be increased in comparison the healthy animals [5,33]. The blood glucose level of rats was increased during diabetes through STZ injection in this study. A decrease has been determined in the blood glucose level of diabetic rats that were given DA1229 as a DPP-4 inhibitor in the 6th week of treatment when compared to diabetic rats [34]. In the study by Zucker diabetic rats showed that sitagliptin reduced blood glucose levels at the end of the 6th week of treatment [35]. In our study, hyperglycemia of diabetic rats was decreased with sitagliptin administration for 15 days. Therefore, we may suggest that sitagliptin is one of the DPP-4 inhibitors that should be administrated on a long-term clinical use. It is suggested that GLP-1 and its analogs may provide beneficial effects for glycemic control and body weight loss [36,37]. Han et al. [38] have reported that the islet size of Zucker diabetic fatty rats treated with sitagliptin is larger than nontreated rats. Furthermore, the area percentage of small Langerhans islets in sitagliptin group is higher than in control Zucker diabetic rats. It has been reported that sitagliptin improves islet morphology in Zucker diabetic rats [39]. In the present study, the percentage of the large Langerhans islets in the diabetic group
was lower than that of the Ctrl rats. This situation can be a result of the STZ toxicity on b cells. The percentage of small islets was the highest in the diabetic + Sit group. For this reason, it is suggested that sitagliptin can induce the formation of new islets. In addition, it has been observed that the morphology of islets in diabetic + Sit group was better than in the diabetic group. Therefore, the degenerated morphology of islets in the diabetic rats was regenerated by means of sitagliptin. The DPP-4 inhibitors have been used in the treatment of type-2 diabetes to stimulate the release of INS, and improve b-cell function and its mass [40]. DPP-4 has the effect stimulating GLP-1 secretion in rats [41]. It is suggested that GLP-1 can stimulate the INS secretion and the release of SS [37,42]. It has been reported that sitagliptin, a DPP-4 inhibitor, increases the active incretin levels in circulation and stimulate the release of INS in type-2 diabetes patients [16,43]. It has been known that SS inhibits insulin and glucagon hormones in pancreas. In our study, the DPP-4 inhibition through sitagliptin increased pancreatic INS, GLU, GLP-1 and GLP-1R expressions in diabetic rats. On the other hand, it slightly reduced SS expression in pancreas. The induced expression of pancreatic insulin and glucagon hormones can be because of reduction of SS hormone. Moreover, the reason of increase in INS expression may be increase the numbers of b cell and/or elongation of b cell life cycle. The increase of insulin
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Fig. 4. Somatostatin (SS) immunopositive cells (arrow) are observed by means of immunohistochemistry in the islets of Langerhans. A, Ctrl; B, diabetic; C, diabetic + Sit groups, n = 8 in each group. Streptavidin–biotin-peroxidase technique, counterstain hematoxylin. Scale bar = 10 mm.
immunopositive cells of pancreas in diabetic rats following sitagliptin could be caused by increased glucagon. It could be suggested that the sitagliptin safeguards against hypoglycemia while it prevents hyperglycemia. It has been known that glucagon and GLP-1 are derived from proglucagon. The provided sitagliptin dose can increase the expression of these hormones through the affected proglucagon synthesis. The DPP-4 inhibition with sitagliptin improved the expression of GLP-1 and GLP-1R in pancreas. Since, GLP-1R stimulates the adenylyl cyclase pathway, GLP-1R increases the insulin synthesis in Langerhans islet and sitagliptin can restore damaged pancreas. There is no consensus about the localization of GLP-1R in pancreas. Various studies have been reported that GLP1R is localized mostly in b cells and to a lesser extent in a and g cells of pancreas islets [44,45]. In the study, we demonstrated for the first time that GLP-1R was localized in both a and b cells of Langerhans islets in newborn type-2 diabetic rats. It was considered that both cells functions could be improved in diabetic rats following treatment with sitagliptin. The changes in all hormones with respect to expression and blood glucose level indicate that sitagliptin may cause the regulation of hyperglycemia and hypoglycemia in type-2 diabetes. Takeda et al. [14] reported in their study on diabetic mice that the DPP-4 inhibition with des-fluoro-sitagliptin attenuates b cell death. Moreover, vildagliptin, a DPP-4 inhibitor, alleviates the destruction of pancreatic b cells in type-1 diabetic rats [46]. The long-term sitagliptin treatment exhibited an antiapoptotic effect on the increased cell death in type-2 diabetic kidneys [47]. On the contrary, the area percentage of apoptotic cells changed in neither diabetic nor control rats in the newborn type-2 diabetic rat model. In this way, no changes were observed on the area percentage of apoptotic cells in islet of Langerhans following treatment with sitagliptin. This situation can be caused by the increased cell regeneration during the growth of newborn rats. At the same time, we can say that the
regeneration in the pancreas of rats may be dominant to over cell death. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidant levels of cells [48]. The increased oxidative stress may have a role in the tissue damage associated with diabetes. The GSH is known to provide a defense against oxidative stress [49]. The serum GSH level did not change in all groups, although it was insignificantly increased in the diabetic + Sit rats. The lipid peroxidation is known as a marker of oxidative stress. It is overproduced in type-2 diabetes and that is related with the oxidative stress. Malondialdehyde (MDA) is one of the most frequently used indicators of lipid peroxidation. It is suggested that vildagliptin reduces the MDA level of pancreatic tissue in adult female diabetic rats [46]. Sitagliptin decreased insignificantly the MDA level of serum in newborn diabetic rats. If the period of sitagliptin treatment is extended, the level of GSH, an antioxidant molecule, can increase in type-2 diabetic rats. Furthermore, higher period of sitagliptin treatment can be effective on the MDA level in serum. In addition, we did not encounter any negative effects of sitagliptin treatment on the oxidative stress. Both SOD and CAT enzymes are antioxidative defense system enzymes. The activity of CAT enzyme was increased while the SOD activity was decreased in the plasma of diabetic rats in the study by Shinde et al. [50]. The CAT and SOD activities increased in diabetic rats by means of vildagliptin treatment [46]. The increased CAT activity in diabetic rats was reduced to a certain extent following sitagliptin treatment. On the contrary, the decreased SOD activity was increased by means of sitagliptin in diabetic rats. In our study, the result of a decreased SOD activity in the diabetes denotes that SOD enzyme can be denatured or inactivated via hyperglycemia-induced an increased ROS. The CAT activity was elevated in the diabetes to eliminate an increased high hydrogen peroxide level. Both of the enzyme activities in diabetes approximated those in Ctrl rats treated with sitagliptin. In that respect, sitagliptin could be used for increased
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Fig. 5. Glucagon-like peptide-1 (GLP-1) immunopositive cells (arrow) are seen (A) Ctrl; (B) diabetic; (C) diabetic + Sit groups and glucagon-like peptide-1 receptor (GLP-1R) immunopositive cells (arrow) in the pancreas; (D) Ctrl; (E) diabetic; (F) diabetic + Sit groups, n = 8 in each group. Streptavidin–biotin-peroxidase technique. Scale bar = 10 mm.
Table 1 Variation in size of islets of Langerhans (%) in control (Ctrl), sitagliptin (Sit), diabetic and diabetic + Sit groups. Islets of Langerhans (%) **
Large Middle** Small** * ** a b c d e f g
Ctrl group* (n = 8) 14.4 0.51 17.5 0.32 68.1 2.04
Sit group* (n = 8) 9.9 0.40 22.6 0.36 67.5 2.04
Diabetic group* (n = 8) a,b
1.6 0.06 9.7 0.25a,d 88.7 2.58b,e
Diabetic + Sit group* (n = 8)
pANOVA
0.3 0.01a,b 4 0.17c 95.7 2.67e,f,g
<0.01 <0.05 <0.001
The sections were stained with six different antibodies in each group. Mean SEM, versus: vs. p < 0.01 vs. Ctrl group. p < 0.01 vs. Sit group. p < 0.05 vs. Ctrl group. p < 0.05 vs. Sit group. p < 0.001 vs. Ctrl group. p < 0.05 vs. diabetic group. p < 0.001 vs. Sit group.
damage resulting from free radicals in diabetes. We can suggest that, the state of oxidative stress in diabetes may moderately be regulated by means of sitagliptin in type-2 diabetic rats.
In conclusion, sitagliptin may be effective in preventing the progression of diabetes and up/down-regulation of glycemia. The increased weight gain in diabetes was decreased following the
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Table 2 The area percentage of insulin (INS) glucagon (GLU), somatostatin (SS), glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-1 receptor (GLP-1R) immunopositive cells and apoptotic cells in pancreas islets of control (Ctrl), sitagliptin (Sit), diabetic and diabetic + Sit rats.
INS (%)* GLU (%)* SS (%)* GLP-1 (%)* GLP-1R (%)* Apoptotic cells (%)*
Ctrl group (n = 8)
Sit group (n = 8)
Diabetic group (n = 8)
Diabetic + Sit group (n = 8)
pANOVA
61 1.0 39 1.0 8 1.0 12 2.0 63 3.0 0
55 3.0 31 1.0a 6 0.9 17 2.0 34 4.0b 0
29 3.0b,d 46 2.0c,d 8 0.7 13 2.0 27 2.0b 0
36 2.0b,d 59 1.0b,d,f 6 0.9 23 2.0a,g 55 2.0e,h 0
<0.001 <0.001 NS <0.05 <0.001 NS
Non-significant: NS, versus: vs. * Mean SEM. a p < 0.01 vs. Ctrl group. b p < 0.001 vs. Ctrl group. c p < 0.05 vs. Ctrl group. d p < 0.001 vs. Sit group. e p < 0.01 vs. Sit group. f p < 0.01 vs. diabetic group. g p < 0.05 vs. diabetic group. h p < 0.001 vs. diabetic group.
Table 3 Glutathione (GSH), malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD) activities in the serum of control (Ctrl), sitagliptin (Sit), diabetic and diabetic + Sit groups.
GSH (nmol/mg protein)* MDA (nmol/mg protein)* CAT (U/mg protein)* SOD (U/mg protein)*
Ctrl group (n = 8)
Sit group (n = 8)
Diabetic group (n = 8)
Diabetic + Sit group (n = 8)
pANOVA
4.70 0.27 0.41 0.64 3.40 0.57 0.82 0.05
5.69 0.32 0.86 0.31 14.27 4.24c 2.25 0.37a
4.43 0.40 0.64 0.13 5.57 5.85b,c 0.47 0.19a,d
5.36 0.55 0.57 0.10 4.99 1.34d 0.72 0.23a,d
NS NS <0.05 <0.001
Non-significant: NS, versus: vs. * Mean SEM. a p < 0.05 vs. Ctrl group. b p < 0.05 vs. Sit group. c p < 0.001 vs. Ctrl group. d p < 0.01 vs. Sit group.
sitagliptin treatment. This agent up-regulates the expressions of GLU, GLP-1, GLP-1R and INS in Langerhans islets hormones. Furthermore, sitagliptin can reduce the diabetes induced oxidative stress state. The results showed that the therapeutic effect of sitagliptin on type-2 diabetes is undeniable. Further studies aimed at investigating the effects of sitagliptin on the apoptotic pathway should provide new clues on the cell death in diabetes. The present study emphasizes that sitagliptin, a DPP-4 inhibitor, has therapeutic properties to a certain extent in type-2 diabetic newborn rats. Conflict of interest The authors declare no conflict of interest. Funding This study was supported by the Scientific Research Projects Coordination Unit of Istanbul University, Project No. 10376. References [1] Havale SH, Pal M. Medicinal chemistry approaches to the inhibition of dipeptidyl peptidase-4 for the treatment of type 2 diabetes. Bioorg Med Chem 2009;17(5):1783–802. [2] Scobie IN. Atlas of diabetes mellitus. 3rd ed. Informa Healthcare; 2007. [3] Robertson RP. Chronic oxidative stress as acentral mechanism for glucosetoxicity in pancreatic islet beta cells in diabetes. J Biol Chem 2004;279(41):42351–54. [4] Bolzan AD, Bianchi MS. Genotoxicity of streptozotocin. Mutat Res 2002; 512(2–3):121–34. [5] Turk N, Dagistanli FK, Sacan O, Yanardag R, Bolkent S. Obestatin and insulin in pancreas of newborn diabetic rats treated with exogenous ghrelin. Acta Histochem 2012;114(4):349–57.
[6] Ma X, Guan Y, Hua X. Glucagon-like peptide 1-potentiated insulin secretion and proliferation of pancreatic b-cells. J Diabetes 2014;6(5):394–402. [7] Mayo KE, Miller LJ, Bataille D, Dalle S, Go¨ke B, Thorens B, et al. International union of pharmacology. XXXV. The glucagon receptor family. Pharmacol Rev 2003;55(1):167–94. [8] Wei Y, Mojsov S. Tissue-specific expression of the human receptor for glucagon-like peptide-I: brain, heart and pancreatic forms have the same deduced amino acid sequences. FEBS Lett 1995;358(3):219–24. [9] Tourrel C, Bailbe D, Meile MJ, Kergoat M, Portha B. Glucagon-like peptide-1 and exendin-4 stimulate b-cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes 2001;50(7):1562–70. [10] Xu G, Stoffers DA, Habener JF, Bonner-Weir S. Exendin-4 stimulates both b-cell replication and neogenesis, resulting in increased b-cell mass and improved glucose tolerance in diabetic rats. Diabetes 1999;48(12):2270–6. [11] Drucker DJ, Nauck MA. The incretin system: glucagon-like peptide-1 receptor agonist and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 2006;368(9548):1696–705. [12] Tahrani AA, Piya MK, Barnett AH. Saxagliptin: a new DPP-4 inhibitor for the treatment of type 2 diabetes mellitus. Adv Ther 2009;26(3):249–62. [13] Riche DM, East HE, Riche KD. Impact of sitagliptin on markers of b-cell function: a meta-analysis. Am J Med Sci 2009;337(5):321–8. [14] Takeda Y, Fujita Y, Honjo J, Yanagimachi T, Sakagami H, Takiyama Y, et al. Reduction of both beta cell death and alpha cell proliferation by dipeptidyl peptidase-4 inhibition in a streptozotocin-induced model of diabetes in mice. Diabetologia 2012;55(2):404–12. [15] Craddy P, Palin HJ, Johnson KI. Comparative effectiveness of dipeptidylpeptidase-4 inhibitors in type 2 diabetes: a systematic review and mixed treatment comparison. Diabetes Ther 2014;5(1):1–41. [16] Subbarayan S, Kipnes M. Sitagliptin: a review. Expert Opin Pharmacother 2011;12(10):1613–22. [17] Meguro S, Kawai T, Matsuhashi T, Sano M, Fukuda K, Itoh H, et al. Basalsupported oral therapy with sitagliptin counteracts rebound hyperglycemia caused by GLP-1 tachyphylaxis. Int J Endocrinol 2014;2014:927317. [18] Shima K, Komatsu M, Noma Y, Miya K. Glycated albumin (GA) is more advantageous than hemoglobin A1c for evaluating the efficacy of sitagliptin in achieving glycemic control in patients with type 2 diabetes. Intern Med 2014;53(8):829–35. [19] Lovshin JA, Drucker DJ. Incretin-based therapies for type 2 diabetes mellitus. Nature 2009;5(5):262–9.
S. Karabulut et al. / Pharmacological Reports 67 (2015) 846–853 [20] Johnson JL, O’Neal KS, Pack CC, Kim MK. Management of a prediabetes case with the DPP-4 inhibitor sitagliptin. Ann Pharmacother 2014;48(6):811–5. [21] Karatug A, Sacan O, Coskun ZM, Bolkent S, Yanardag R, Turk N, et al. Regulation of gene expression and biochemical changes in small intestine of newborn diabetic rats by exogenous ghrelin. Peptides 2012;33(1):101–8. [22] Bonner-Weir S, Trent DF, Honey RN, Weir GC. Responses of neonatal rat islets to streptozotocin: limited B-cell regeneration and hyperglycemia. Diabetes 1981;30(1):64–9. [23] Thyssen S, Arany E, Hill DJ. Ontogeny of regeneration of b-cells in the neonatal rat after treatment with streptozotocin. Endocrinology 2006;147(5):2346–56. [24] Beutler E. Red cell metabolism: a manual of biochemical methods. London: Academic Press; 1971. [25] Ledwozyw A, Michalak J, Stepien´ A, Kadziołka A. The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986;155(3):275–83. [26] Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121–6. [27] Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44(1):276–87. [28] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193(1):265–75. [29] Goncalves A, Leal L, Paiva A, Lemos ET, Teixeira F, Ribeiro CF. Protective effects of the dipeptidyl peptidase IV inhibitor sitagliptin in the blood–retinal barrier in a type 2 diabetes animal model. Diabetes Obes Metab 2012;14(5):454–63. [30] Mu J, Woods J, Zhou YP. Chronic inhibition of dipeptidyl peptidase-4 with a sitagliptin analog preserves pancreatic beta-cell mass and function in a rodent model of type 2 diabetes. Diabetes 2006;55(6):1695–704. [31] Kim SJ, Nian C, Doudet DJ, McIntosh CHS. Inhibition of dipeptidyl peptidase IV with sitagliptin (MK0431) prolongs islet graft survival in streptozotocininduced diabetic mice. Diabetes 2008;57(5):1331–9. [32] Sharma AK, Sharma A, Kumari R, Kishore K, Sharma D, Srinivasan BP, et al. Sitagliptin, sitagliptin and metformin, or sitagliptin and amitriptyline attenuate streptozotocin–nicotinamide induced diabetic neuropathy in rats. J Biomed Res 2012;26(3):200–10. [33] Coskun ZM, Bolkent S. Biochemical and immunohistochemical changes in delta-9 tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochem 2014;116(1):112–6. [34] Cho JM, Jang HW, Cheon H, Jeong YT, Kim DH, Lim YM. A novel dipeptidyl peptidase IV inhibitor DA-1229 ameliorates streptozotocin-induced diabetes by increasing b-cell replication and neogenesis. Diabetes Res Clin Pract 2011;91(1):72–9. [35] Mega C, de Lemos ET, Vala H, Fernandes R, Oliveira J, Mascarenhas-Melo F, et al. Diabetic nephropathy amelioration by a low-dose sitagliptin in an animal model of type 2 diabetes (Zucker diabetic fatty rat). Exp Diabetes Res 2011;2011:162092.
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Immunohistochemical, apoptotic and biochemical changes by dipeptidyl peptidase-4 inhibitor-sitagliptin in type-2 diabetic rats Sezin Karabulut a, Zeynep Mine Coskun b, Sema Bolkent a,* a b
Department of Medical Biology, Faculty of Cerrahpasa Medicine, Istanbul University, Istanbul, Turkey Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Istanbul Bilim University, Istanbul, Turkey
A R T I C L E I N F O
Article history: Received 18 September 2014 Received in revised form 26 November 2014 Accepted 19 January 2015 Available online 1 February 2015 Keywords: Apoptosis Rat Sitagliptin Type-2 diabetes Pancreas
A B S T R A C T
Background: Diabetes is a major public health problem that is rapidly increasing in prevalence. In this study, the effects of sitagliptin, a dipeptidyl peptidase-4 inhibitor, were examined on newborn diabetic rat model. Methods: Wistar albino newborn rats were divided into control (Ctrl), sitagliptin (Sit), diabetic and diabetic + Sit groups. On the second day after the birth, 100 mg/kg streptozotocin (STZ) was administered intraperitoneally in a single dose to induce type-2 diabetes in rats. The Sit and diabetic + Sit groups were administered sitagliptin (1.5 mg/kg subcutaneous) as of the day 5 for 15 days. The pancreas sections were stained with insulin (INS), glucagon (GLU), somatostatin (SS), glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-1 receptor (GLP-1R) antibodies by the streptavidin–biotin peroxidase technique. The TUNEL method for apoptosis and biochemical analysis were performed in the pancreas and serum, respectively. Results: Body weight and blood glucose levels showed significant differences among all groups on days 11 and 20. In diabetic rats following treatment with sitagliptin, the area percentage of INS immunopositive cells increased while the area percentage of SS immunopositive cells decreased, insignificantly. A significant increase was observed on the area percentage of GLU, GLP-1 and GLP-1R immunopositive cells in the diabetic + Sit group when compared to the diabetic group. The area percentage of apoptotic cells was the same among all groups. While serum glutathione and malondialdehyde levels demonstrated insignificant alterations, the catalase and superoxide dismutase activity significantly changed among four groups. Conclusion: According to our findings, sitagliptin may be a useful therapeutic agent to a certain extent of type-2 diabetic condition. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction Diabetes mellitus is one of the most common metabolic diseases in the world that is characterized by increased blood glucose level during fasting or oral glucose tolerance test [1,2]. Type-2 diabetes is identified by means of impairments in the b-cell function and insulin (INS) secretion [1,3]. Streptozotocin (STZ) is widely preferred in investigations as part of studies of the type-2 diabetic model [4]. In animal models, STZ is administered to newborn rats to induce type-2 diabetes [5].
* Corresponding author. E-mail addresses: [email protected], [email protected] (S. Bolkent).
Incretin hormones are important to understand the mechanism of glucose homeostasis. Glucagon-like peptide-1 (GLP-1), one of the most important of incretin hormones, increases insulin secretion after food ingestion [6]. It is suggested that GLP-1 receptor (GLP-1R) is expressed on a and b cells of pancreas, gastrointestinal system, kidney, lung, heart and brain [7,8]. It has been reported that GLP-1 provides b cell regeneration, differentiation and prevents apoptosis via the activation of GLP-1R [9,10]. GLP-1 hormones are degraded by the dipeptidyl peptidase-4 (DPP-4) enzyme [11]. DPP-4 is expressed in pancreas, brain, lung, kidney, liver, intestine, adrenal gland and lymphocytes [12]. Long-term studies suggest that DPP-4 inhibitors decreased b cell death, increase its neogenesis and function in diabetic animals [13,14]. Sitagliptin, a novel DPP-4 inhibitor, is a therapeutic agent for type-2 diabetes that is administered either as a monotherapy or
http://dx.doi.org/10.1016/j.pharep.2015.01.010 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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combination therapy [15,16]. Furthermore, the agent provides improvements in glycemic control [17]. DPP-4 inhibitors-related studies on humans have found a decrease in HbA1c levels [18]. It has been reported that DPP-4 inhibitors do not affect body weight [19]. Moreover, it is suggested that sitagliptin may have a role as a treatment agent for pre-diabetes [20]. In our study, we aimed to explore the biochemical alterations in serum, INS, glucagon (GLU), somatostatin (SS), GLP-1 and GLP-1R expressions and programmed cell death (apoptosis) in the pancreas of healthy and type-2 diabetic rats following sitagliptin administration.
sections were prepared by means of substituting antibodies with phosphate buffer saline (PBS). The rat stomach sections were used as SS positive controls and non-experimental rat pancreas sections were used as INS, GLU, GLP-1 and GLP-1R positive controls because of their high expressions. The photographs of the Langerhans islets in pancreas were taken and the size of islets calculated using a Nikon Eclipse 80i light microscope equipped with a digital camera using NIS-Elements-D 3.1 microscope imaging software program.
Materials and methods
The apoptosis was determined by the terminal deoxynucleotidyl transferase (Tdt) mediated dUTP (TUNEL) method (APOPTAG1 kit; Millipore S7101) according to the manufacturer’s instructions. The reaction was observed using an AEC substrate kit. The negative control sections were prepared by substituting Tdt enzyme with distilled water. The rat breast tissue sections (S7115) were used as positive controls for the detection of programmed cell death.
Animals and tissue preparation
Apoptosis
All animal experimental procedures were approved by the Istanbul University Local Ethics Committee on Animal Research. When the newborn Wistar albino rats were 48-h-old following birth, they were included in the experimental groups. There were four groups of newborn rats. Group I (n = 8): the control group (Ctrl) consisted of newborn rats that were given physiological saline for a period of 18 days, intraperitoneally (ip). Group II (n = 8): the sitagliptin group (Sit) was treated with 1.5 mg/kg/day sitagliptin (Januvia, Merck) dissolved in physiological saline from the fifth day for a period of 15 days, subcutaneously (sc). Group III (n = 8): for streptozotocin (STZ)-induced diabetic rats (diabetic), 100 mg/kg STZ (Sigma–Aldrich, S0130) was dissolved in physiological saline and given as a single intraperitoneal dose on the second day following birth [21,22]. The rats whose blood glucose levels were 200 mg/dl or more on the second day following STZ injection were considered as diabetic. Group IV (n = 8): the diabetic group which was given sitagliptin (diabetic + Sit) on the fifth day following birth for a period of 15 days. The blood glucose levels of animals in all groups were measured with a glucometer (Accu-check, Roche Diagnostics GmbH) using the blood samples collected from the tail vein on days 2, 4, 11 and 20. The body weights of the animals were also measured at the same time intervals. On the 20th day, the animals were left to fast for 1 h; blood and pancreatic tissue samples were collected under ketamin-HCl (50 mg/kg) anesthesia. The tissue samples were fixed in 10% neutral buffered formalin for 24 h at +4 8C and then embedded in paraffin using routine light microscopy processing methods.
The blood samples were then centrifuged at 3000 rpm for 10 min to separate the serum. The biochemical parameters were determined on serum samples were frozen at 80 8C. Serum glutathione (GSH), malondialdehyde (MDA) levels, catalase (CAT) and superoxide dismutase (SOD) activities were measured. GSH levels were determined according to Beutler’s method using metaphosphoric acid for protein precipitation and 5,50 -dithiobis (2-nitro benzoic acid (Sigma–Aldrich) for the color development at 412 nm [24]. The MDA levels were estimated by using the Ledwozyw’s methods [25]. The serum samples were mixed thoroughly with a solution of trichloroacetic acid (TCA) (Sigma– Aldrich) (30%), TBA (Merck) (0.75%) and 5 M hydrochloric acid. The samples were measured at 535 nm. The CAT activity was assayed by the methods of Aebi. The reaction mixture was made up of the sample, 50 mM phosphate buffer pH 7.0 and 10 mM H2O2. The reduction rate of H2O2 was observed to be 240 nm at the room temperature for 60 s [26]. The SOD activity was determined by the method developed by Beauchamp and Fridovich that involves the inhibition of nitroblue tetrazolium (NBT) (Sigma–Aldrich, Louis) [27]. The protein content in the serum was estimated by the method of Lowry [28].
The study of Langerhans islet size and immunohistochemical staining
Statistical analysis
The sections of pancreases were stained by using hematoxylin and eosin. All of the Langerhans islets in pancreas were classified as small, medium and large according to their size after calculating the areas. Large islets were >10,000 m2, medium islets were >5000–10,000 m2, and small islets were <5000 mm2 [23]. For immunohistochemical staining, the sections were incubated with mouse monoclonal INS (dilution 1:1000; Sigma–Aldrich, I2018), mouse monoclonal GLU (dilution 1:2000; Sigma–Aldrich, G2654) and rabbit polyclonal SS (dilution 1:2500; Chemicon, AB1976), mouse monoclonal GLP-1 (dilution 1:500; Santa Cruz, sc57166) and rabbit polyclonal GLP-1R (dilution 1:50; Novus, NLS1205) antibodies. They were stained with the streptavidin– biotin-peroxidase method using histostain-plus bulk kit (Zymed 85-9043). The detection procedures were carried out as described by the manufacturer. The enzyme activity was developed using 3-amino-9-ethyl-carbazole (AEC) substrate kit (Zymed 00-2007) and the sections were then counterstained with Mayer’s hematoxylin. The staining intensity was scored from one to three as weak (+), medium (++) and strong (+++). The negative control
The INS, GLU, SS, GLP-1, GLP-1R immunopositive cells and apoptotic cells were counted in all islets of Langerhans for each section of rat pancreas. The area percentages of immunopositive and apoptotic cells in the islets were calculated by using the formula of (labeling area/total area) 100. The values obtained were evaluated statistically. SPSS 21.0 software was used for the statistical analysis. The results were expressed as means SEM. The statistical evaluations were performed using Kruskal–Wallis test followed by Mann– Whitney U test. A p-value <0.05 was considered to be significant.
Biochemical assay
Results Body weight and blood glucose In Figs. 1 and 2, the changes in the comparable body weight and blood glucose levels for all experimental groups are presented. A significant difference in body weight and blood glucose level was
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Body weight (g)
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Fig. 1. The changes of body weights (g) in experimental rats on days 2, 4, 11 and 20, n = 8 in each group. Data are expressed as means SEM. ap < 0.001 vs. control (Ctrl) group, bp < 0.01 vs. Ctrl group, cp < 0.001 vs. sitagliptin (Sit) group. Versus: vs.
Blood Glucose (mg/dl)
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immunopositive islet area percentage increased significantly in the diabetic group as compared to Ctrl group (p < 0.05). The GLU area percentage of the islet significantly increased in the diabetic + Sit group as compared to the diabetic group (p < 0.01) (Fig. 3). There was no significant difference among four groups with respect to the area percentage of SS immunopositive cells (Fig. 4). The immunopositive cells area percentage of GLP-1 in the islets was almost the same in the diabetic group as compared to the Ctrl group. The area percent of GLP-1 in the islet significantly increased in the diabetic + Sit group when compared to diabetic (p < 0.05) (Fig. 5). The GLP-1 immunopositive islet area significantly decreased in the Sit and diabetic groups as compared to Ctrl group, respectively (p < 0.001, p < 0.001). The GLP-1R percentage in the islet area was significantly increased in the diabetic + Sit group when compared to diabetic (p < 0.001). The GLP-1 peptide was located in islet periphery, while the GLP-1R was located both center and periphery of islet (Fig. 5). With respect to reaction intensity, the staining intensity of insulin immunopositive cells in diabetic + Sit group (++) was weaker than the other groups (+++). However, the staining intensity of glucagon (++), somatostatin (++), glucagon-like peptide-1 (+) and glucagon-like peptide-1 receptor (+) immunopositive cells and apoptotic cells (+++) did not vary between diabetic and diabetic + Sit groups. The programmed cell death by means of TUNEL method
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Fig. 2. Effects of sitagliptin on blood glucose (mg/dl) levels in rats on days 2, 4, 11 and 20, n = 8 in each group. Data are expressed as means SEM. ap < 0.01 vs. Ctrl group, bp < 0.001 vs. sitagliptin (Sit) group, cp < 0.001 vs. the Ctrl group. Versus: vs.
determined among all groups, respectively (pANOVA < 0.001, pANOVA < 0.001). There was a significant increase in body weights of diabetic group as compared to Ctrl group on days 4, 11 and 20, respectively (p < 0.001, p < 0.01, p < 0.001). However, it was observed that the body weight of the diabetic + Sit group decreased insignificantly when compared to diabetic rats on days 11 and 20. There was a significant increase in the blood glucose levels of diabetic rats as compared to Ctrl rats on days 4, 11 and 20, respectively (p < 0.001, p < 0.01, p < 0.001). It was determined that there were no changes in the blood glucose levels of diabetic + Sit group as compared to diabetic group on days 11 and 20. The results of Langerhans islet size measurement and immunohistochemistry The pancreas sections from Ctrl, diabetic and diabetic + Sit rats are shown in Figs. 3–5, respectively. It was seen that animals in the Ctrl group had the greatest area percent of large size islets as compared to the other groups. Diabetic + Sit rats had the highest percentage of small size islets when compared to the other groups. It was observed that diabetic + Sit group had more small size islets when compared to diabetic group (Table 1). The area percentage of INS, GLU, SS, GLP-1 and GLP-1R immunopositive cells for each group is presented in Table 2, respectively (pANOVA < 0.001, pANOVA < 0.001, pANOVA > 0.05, pANOVA < 0.05, pANOVA < 0.001). The area percentage of INS immunopositive cells significantly decreased in the diabetic group as compared to Ctrl group (p < 0.001). The INS area percentage in the islet of Langerhans increased insignificantly in the diabetic + Sit group as compared to diabetic rats (Fig. 3). The GLU
The TUNEL method was applied to pancreas sections with stained nucleus were evaluated as apoptotic cells (data not shown). The apoptotic cells percentage did not demonstrate a significant difference among four groups. It was evaluated that the reaction intensity of apoptotic cells was the same in islets of rats (+++). Biochemical results The effect of sitagliptin on GSH, MDA levels and CAT, SOD activities in serum of diabetic and Ctrl rats are seen in Table 3. The GSH level showed an insignificant increase in diabetic rats treated with sitagliptin as compared to other diabetic rats. There were no significant differences among all groups in terms of MDA levels. The MDA level showed an insignificant decrease in the diabetic + sitagliptin group as compared to the diabetic group. The CAT and SOD activity significantly varied among all groups, respectively (pANOVA < 0.05, pANOVA < 0.001). The CAT activity was found to be insignificantly increased in diabetic rats as compared to the Ctrl group. On the contrary, SOD activity showed a significant decrease in the diabetic group when compared to the Ctrl group (p < 0.05). Administration of sitagliptin to diabetic rats insignificantly decreased CAT activity and increased the SOD activity as compared to diabetic rats. Discussion The studies show that a decrease has been observed in body weights of diabetic adult rats as compared with healthy rats [29,30]. Conversely, it is suggested that there are no differences in body weight between healthy and diabetic rats at the end of third week after birth [5]. It is reported that sitagliptin did not change the body weights of different species of diabetic animals [31,32]. By contrast with the previous studies, administration of sitagliptin regulated the body weight of diabetic rats in this study. There was an extreme increase in body weight of diabetic rats on days 11 and 20. The body weight in diabetic rats approached the body weight of normal rats on days 11 and 20 with administration of sitagliptin. This difference supports the view that sitagliptin can
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Fig. 3. Insulin (INS) and glucagon (GLU) immunopositive cells (arrow) are observed by means of immunohistochemistry in the islets of Langerhans, n = 8 in each group. Streptavidin–biotin-peroxidase technique, counterstain hematoxylin. Scale bar = 10 mm.
be effective on the regulation of body weight in type-2 diabetic rats. The blood glucose level of both diabetic newborn and adult rats has been shown to be increased in comparison the healthy animals [5,33]. The blood glucose level of rats was increased during diabetes through STZ injection in this study. A decrease has been determined in the blood glucose level of diabetic rats that were given DA1229 as a DPP-4 inhibitor in the 6th week of treatment when compared to diabetic rats [34]. In the study by Zucker diabetic rats showed that sitagliptin reduced blood glucose levels at the end of the 6th week of treatment [35]. In our study, hyperglycemia of diabetic rats was decreased with sitagliptin administration for 15 days. Therefore, we may suggest that sitagliptin is one of the DPP-4 inhibitors that should be administrated on a long-term clinical use. It is suggested that GLP-1 and its analogs may provide beneficial effects for glycemic control and body weight loss [36,37]. Han et al. [38] have reported that the islet size of Zucker diabetic fatty rats treated with sitagliptin is larger than nontreated rats. Furthermore, the area percentage of small Langerhans islets in sitagliptin group is higher than in control Zucker diabetic rats. It has been reported that sitagliptin improves islet morphology in Zucker diabetic rats [39]. In the present study, the percentage of the large Langerhans islets in the diabetic group
was lower than that of the Ctrl rats. This situation can be a result of the STZ toxicity on b cells. The percentage of small islets was the highest in the diabetic + Sit group. For this reason, it is suggested that sitagliptin can induce the formation of new islets. In addition, it has been observed that the morphology of islets in diabetic + Sit group was better than in the diabetic group. Therefore, the degenerated morphology of islets in the diabetic rats was regenerated by means of sitagliptin. The DPP-4 inhibitors have been used in the treatment of type-2 diabetes to stimulate the release of INS, and improve b-cell function and its mass [40]. DPP-4 has the effect stimulating GLP-1 secretion in rats [41]. It is suggested that GLP-1 can stimulate the INS secretion and the release of SS [37,42]. It has been reported that sitagliptin, a DPP-4 inhibitor, increases the active incretin levels in circulation and stimulate the release of INS in type-2 diabetes patients [16,43]. It has been known that SS inhibits insulin and glucagon hormones in pancreas. In our study, the DPP-4 inhibition through sitagliptin increased pancreatic INS, GLU, GLP-1 and GLP-1R expressions in diabetic rats. On the other hand, it slightly reduced SS expression in pancreas. The induced expression of pancreatic insulin and glucagon hormones can be because of reduction of SS hormone. Moreover, the reason of increase in INS expression may be increase the numbers of b cell and/or elongation of b cell life cycle. The increase of insulin
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Fig. 4. Somatostatin (SS) immunopositive cells (arrow) are observed by means of immunohistochemistry in the islets of Langerhans. A, Ctrl; B, diabetic; C, diabetic + Sit groups, n = 8 in each group. Streptavidin–biotin-peroxidase technique, counterstain hematoxylin. Scale bar = 10 mm.
immunopositive cells of pancreas in diabetic rats following sitagliptin could be caused by increased glucagon. It could be suggested that the sitagliptin safeguards against hypoglycemia while it prevents hyperglycemia. It has been known that glucagon and GLP-1 are derived from proglucagon. The provided sitagliptin dose can increase the expression of these hormones through the affected proglucagon synthesis. The DPP-4 inhibition with sitagliptin improved the expression of GLP-1 and GLP-1R in pancreas. Since, GLP-1R stimulates the adenylyl cyclase pathway, GLP-1R increases the insulin synthesis in Langerhans islet and sitagliptin can restore damaged pancreas. There is no consensus about the localization of GLP-1R in pancreas. Various studies have been reported that GLP1R is localized mostly in b cells and to a lesser extent in a and g cells of pancreas islets [44,45]. In the study, we demonstrated for the first time that GLP-1R was localized in both a and b cells of Langerhans islets in newborn type-2 diabetic rats. It was considered that both cells functions could be improved in diabetic rats following treatment with sitagliptin. The changes in all hormones with respect to expression and blood glucose level indicate that sitagliptin may cause the regulation of hyperglycemia and hypoglycemia in type-2 diabetes. Takeda et al. [14] reported in their study on diabetic mice that the DPP-4 inhibition with des-fluoro-sitagliptin attenuates b cell death. Moreover, vildagliptin, a DPP-4 inhibitor, alleviates the destruction of pancreatic b cells in type-1 diabetic rats [46]. The long-term sitagliptin treatment exhibited an antiapoptotic effect on the increased cell death in type-2 diabetic kidneys [47]. On the contrary, the area percentage of apoptotic cells changed in neither diabetic nor control rats in the newborn type-2 diabetic rat model. In this way, no changes were observed on the area percentage of apoptotic cells in islet of Langerhans following treatment with sitagliptin. This situation can be caused by the increased cell regeneration during the growth of newborn rats. At the same time, we can say that the
regeneration in the pancreas of rats may be dominant to over cell death. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidant levels of cells [48]. The increased oxidative stress may have a role in the tissue damage associated with diabetes. The GSH is known to provide a defense against oxidative stress [49]. The serum GSH level did not change in all groups, although it was insignificantly increased in the diabetic + Sit rats. The lipid peroxidation is known as a marker of oxidative stress. It is overproduced in type-2 diabetes and that is related with the oxidative stress. Malondialdehyde (MDA) is one of the most frequently used indicators of lipid peroxidation. It is suggested that vildagliptin reduces the MDA level of pancreatic tissue in adult female diabetic rats [46]. Sitagliptin decreased insignificantly the MDA level of serum in newborn diabetic rats. If the period of sitagliptin treatment is extended, the level of GSH, an antioxidant molecule, can increase in type-2 diabetic rats. Furthermore, higher period of sitagliptin treatment can be effective on the MDA level in serum. In addition, we did not encounter any negative effects of sitagliptin treatment on the oxidative stress. Both SOD and CAT enzymes are antioxidative defense system enzymes. The activity of CAT enzyme was increased while the SOD activity was decreased in the plasma of diabetic rats in the study by Shinde et al. [50]. The CAT and SOD activities increased in diabetic rats by means of vildagliptin treatment [46]. The increased CAT activity in diabetic rats was reduced to a certain extent following sitagliptin treatment. On the contrary, the decreased SOD activity was increased by means of sitagliptin in diabetic rats. In our study, the result of a decreased SOD activity in the diabetes denotes that SOD enzyme can be denatured or inactivated via hyperglycemia-induced an increased ROS. The CAT activity was elevated in the diabetes to eliminate an increased high hydrogen peroxide level. Both of the enzyme activities in diabetes approximated those in Ctrl rats treated with sitagliptin. In that respect, sitagliptin could be used for increased
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Fig. 5. Glucagon-like peptide-1 (GLP-1) immunopositive cells (arrow) are seen (A) Ctrl; (B) diabetic; (C) diabetic + Sit groups and glucagon-like peptide-1 receptor (GLP-1R) immunopositive cells (arrow) in the pancreas; (D) Ctrl; (E) diabetic; (F) diabetic + Sit groups, n = 8 in each group. Streptavidin–biotin-peroxidase technique. Scale bar = 10 mm.
Table 1 Variation in size of islets of Langerhans (%) in control (Ctrl), sitagliptin (Sit), diabetic and diabetic + Sit groups. Islets of Langerhans (%) **
Large Middle** Small** * ** a b c d e f g
Ctrl group* (n = 8) 14.4 0.51 17.5 0.32 68.1 2.04
Sit group* (n = 8) 9.9 0.40 22.6 0.36 67.5 2.04
Diabetic group* (n = 8) a,b
1.6 0.06 9.7 0.25a,d 88.7 2.58b,e
Diabetic + Sit group* (n = 8)
pANOVA
0.3 0.01a,b 4 0.17c 95.7 2.67e,f,g
<0.01 <0.05 <0.001
The sections were stained with six different antibodies in each group. Mean SEM, versus: vs. p < 0.01 vs. Ctrl group. p < 0.01 vs. Sit group. p < 0.05 vs. Ctrl group. p < 0.05 vs. Sit group. p < 0.001 vs. Ctrl group. p < 0.05 vs. diabetic group. p < 0.001 vs. Sit group.
damage resulting from free radicals in diabetes. We can suggest that, the state of oxidative stress in diabetes may moderately be regulated by means of sitagliptin in type-2 diabetic rats.
In conclusion, sitagliptin may be effective in preventing the progression of diabetes and up/down-regulation of glycemia. The increased weight gain in diabetes was decreased following the
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Table 2 The area percentage of insulin (INS) glucagon (GLU), somatostatin (SS), glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-1 receptor (GLP-1R) immunopositive cells and apoptotic cells in pancreas islets of control (Ctrl), sitagliptin (Sit), diabetic and diabetic + Sit rats.
INS (%)* GLU (%)* SS (%)* GLP-1 (%)* GLP-1R (%)* Apoptotic cells (%)*
Ctrl group (n = 8)
Sit group (n = 8)
Diabetic group (n = 8)
Diabetic + Sit group (n = 8)
pANOVA
61 1.0 39 1.0 8 1.0 12 2.0 63 3.0 0
55 3.0 31 1.0a 6 0.9 17 2.0 34 4.0b 0
29 3.0b,d 46 2.0c,d 8 0.7 13 2.0 27 2.0b 0
36 2.0b,d 59 1.0b,d,f 6 0.9 23 2.0a,g 55 2.0e,h 0
<0.001 <0.001 NS <0.05 <0.001 NS
Non-significant: NS, versus: vs. * Mean SEM. a p < 0.01 vs. Ctrl group. b p < 0.001 vs. Ctrl group. c p < 0.05 vs. Ctrl group. d p < 0.001 vs. Sit group. e p < 0.01 vs. Sit group. f p < 0.01 vs. diabetic group. g p < 0.05 vs. diabetic group. h p < 0.001 vs. diabetic group.
Table 3 Glutathione (GSH), malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD) activities in the serum of control (Ctrl), sitagliptin (Sit), diabetic and diabetic + Sit groups.
GSH (nmol/mg protein)* MDA (nmol/mg protein)* CAT (U/mg protein)* SOD (U/mg protein)*
Ctrl group (n = 8)
Sit group (n = 8)
Diabetic group (n = 8)
Diabetic + Sit group (n = 8)
pANOVA
4.70 0.27 0.41 0.64 3.40 0.57 0.82 0.05
5.69 0.32 0.86 0.31 14.27 4.24c 2.25 0.37a
4.43 0.40 0.64 0.13 5.57 5.85b,c 0.47 0.19a,d
5.36 0.55 0.57 0.10 4.99 1.34d 0.72 0.23a,d
NS NS <0.05 <0.001
Non-significant: NS, versus: vs. * Mean SEM. a p < 0.05 vs. Ctrl group. b p < 0.05 vs. Sit group. c p < 0.001 vs. Ctrl group. d p < 0.01 vs. Sit group.
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