The Biological Impacts of Sitagliptin on the Pancreas of a Rat Model of Type 2 Diabetes Mellitus: Drug Interactions with Metformin

Abstract

Sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, is a beneficial class of antidiabetic drugs. However, a major debate about the risk of developing pancreatitis is still existing. The aim of the work was to study the histological and immunohistochemical effects of sitagliptin on both endocrine and exocrine pancreases in a rat model of type 2 diabetes mellitus and to correlate these effects with the biochemical findings. Moreover, a possible synergistic effect of sitagliptin, in combination with metformin, was also evaluated. Fifty adult male rats were used and assigned into five equal groups. Group 1 served as control. Group 2 comprised of untreated diabetic rats. Group 3 diabetic rats received sitagliptin. Group 4 diabetic rats received metformin. Group 5 diabetic rats received both combined. Treatments were given for 4 weeks after the induction of diabetes. Blood samples were collected for biochemical assay before the sacrification of rats. Pancreases were removed, weighed, and were processed for histological and immunohistochemical examination. In the untreated diabetic group, the islets appeared shrunken with disturbed architecture and abnormal immunohistochemical reactions for insulin, caspase-3, and inducible nitric oxide synthase (iNOS). The biochemical findings were also disturbed. Morphometrically, there was a significant decrease in the islet size and islet number. Treatment with sitagliptin, metformin, and their combination showed an improvement, with the best response in the combined approach. No evidence of pancreatic injury was identified in the sitagliptin-treated groups. In conclusion, sitagliptin had a cytoprotective effect on beta-cell damage. Furthermore, the data didn't indicate any detrimental effects of sitagliptin on the exocrine pancreas.

Keywords: HFD/STZ diabetes; caspase-3; iNOS; immunohistochemistry; insulin; pancreas; rat; sitagliptin.

Figure 1

Changes in serum glucose (mg/dL) during the treatment period in the different studied groups. The values were expressed as mean ± SD (n = 10). ^a: significantly different, compared to the control group. ^b: significantly different, compared to the untreated diabetic group. ^c: significantly different, compared to the sitagliptin and metformin-treated groups, using post hoc ANOVA (LSD), p < 0.05.

Figure 2

Changes in serum insulin and HOMA-IR in the different studied groups at the end of the treatment period. The values were expressed as mean ± SD (n = 10). ^a: significantly different, compared to the control group. ^b: significantly different, compared to the diabetic untreated group. ^c: significantly different, compared to the sitagliptin and metformin-treated groups. ^d: significantly different, compared to metformin, using post hoc ANOVA (LSD), p < 0.05.

Figure 3

Changes in the blood glucose level (mg/dL) during the conduction of OGTT (oral glucose tolerance test) in the experimental groups at the end of the treatment period. The data were expressed as a mean ± SD (n = 10). ^a: significantly different, compared to the control group. ^b: significantly different, compared to the untreated diabetic group. ^c: significantly different, compared to the sitagliptin and metformin-treated groups, using post hoc ANOVA (LSD), p < 0.05.

Figure 4

H&E stained microscopic photomicrographs of the different studied groups. (A). Control rats show the endocrine pancreas (EN) with histologic features of normal islets. The exocrine pancreatic acini (EX) appear with normal pyramidal lining cells, showing basal basophilia and apical acidophilia. (B,C). The untreated diabetic group shows shrunken islets with a drastic decrease in the number of their cells. Some islet cells are distorted with vacuolated cytoplasm (stars), small-sized darkly stained nuclei (arrows), and congested blood capillaries (double arrows) in-between. (D). The sitagliptin-treated group shows an apparent increase in islet size and normal histoarchitecture. Few islet cells show small, deeply stained nuclei (arrowheads). Pancreatic acini appear closed packed with normal histology (E). Metformin-treated group shows an apparent improvement in the islet size, normal cellular arrangement. Apoptotic changes, in the form of small pyknotic nuclei and deeply acidophilic cytoplasm (arrowheads), are seen. (F). The combined therapy group shows improved islet size, histoarchitecture, and cytology. Apoptotic nuclei (arrowheads) are also seen. The pancreatic acini appear normal. Magnification: (A,F) ×200, (B–E) ×400.

Figure 5

Insulin immunostained photomicrographs of pancreatic islets of the different groups. Arrows mark the brown coloration that is indicative of the positive cytoplasmic reaction. A marked positive reaction is marked in the control group (A), in the islet beta cells and surrounded by a peripheral mantle zone of negatively immunostained cells (arrowheads). The shrunken islet, in the untreated diabetic group (B), shows a less positive reaction. Sitagliptin-treated (C), metformin-treated (D), and both-treated (E) groups show viable degrees of improvement of insulin immunoreactivity, which is marked in the combined therapy group. Magnification: (A–E) ×400.

Figure 6

Caspase-3 immunostained photomicrographs of pancreatic islets of the different groups. Arrows mark the brown coloration that is indicative of the positive cytoplasmic reaction. Caspase-3 is not detected in the islet cells of the control rats (A). Markedly positive cytoplasmic immunoreaction is noted in the islet cells of the untreated diabetic group (B). Treated groups (C–E) show a decrease in the caspase-3 immunoreaction, with the least reaction in the combined therapy group (E). Magnification: (A–E) ×400.

Figure 7

iNOS immunostained photomicrographs of pancreatic sections of the different studied groups. Arrows mark the brown coloration that is indicative of the positive cytoplasmic reaction. The control group (A) shows a negative reaction. The iNOS immunoreaction is limited to the islet cells of the untreated diabetic group (B), with no reaction detected in the pancreatic acini. All treated groups (C–E) show negative iNOS immunoreaction in both endocrine and exocrine pancreases. Magnification: (A–D) ×400 and (E) ×200.

Figure 8

The pancreatic islet number (A) and islet size (B) in the different studied groups, at the end of the study period. The data were expressed as mean ± SD (n = 10). ^a: significantly different when compared to control, ^b: significantly different when compared to diabetic, ^c: significantly different when compared to sitagliptin and metformin, ^d: compared to metformin, using post hoc ANOVA (LSD), p < 0.05.

Figure 9

The area percent of insulin (A), caspase-3 (B), and iNOS (C) immunoreactivity in the different studied groups, at the end of the study period. The data were expressed as mean ± SD (n = 10). ^a: significantly different when compared to control, ^b: significantly different when compared to diabetic, ^c: significantly different when compared to sitagliptin and metformin, ^d: compared to metformin, using post hoc ANOVA (LSD), p < 0.05.