Sitagliptin prevents aggravation of endocrine and exocrine pancreatic damage in the Zucker Diabetic Fatty rat - focus on amelioration of metabolic profile and tissue cytoprotective properties

Abstract

Background: The purpose of this study was to investigate some of the possible mechanisms underlying the protective effects of a dipeptidyl peptidase IV (DPP-IV) inhibitor, sitagliptin, on pancreatic tissue in an animal model of type 2 diabetes mellitus (T2DM), the Zucker Diabetic Fatty (ZDF) rat, focusing on glycaemic, insulinic and lipidic profiles, as well as, on apoptosis, inflammation, angiogenesis and proliferation mediators.

Methods: Male obese diabetic ZDF (fa/fa) rats, aged 20 weeks, were treated with sitagliptin (10 mg/kg bw/day) during 6 weeks and compared to untreated diabetic and lean control littermates. Metabolic data was evaluated at the beginning and at the end of the treatment, including glycaemia, HbA1c, insulinaemia, HOMA-beta and TGs. Endocrine and exocrine pancreas lesions were assessed semiquantitatively by histopathological methods. Pancreas gene (mRNA) and protein expression of mediators of apoptotic machinery, inflammation and angiogenesis/proliferation (Bax, Bcl2, IL-1β, VEGF, PCNA and TRIB3) were analyzed by RT-qPCR and/or by immunohistochemistry.

Results: Sitagliptin treatment for 6 weeks (between 20 and 26 week-old) was able to significantly (p < 0.001) ameliorate all the metabolic parameters, by preventing the increase in blood glucose and in serum TGs contents (16.54% and 37.63%, respectively, vs untreated), as well as, by preventing the decrease in serum insulin levels and in the functional beta cells capacity accessed via HOMA-beta index (156.28% and 191.74%, respectively, vs untreated). Sitagliptin-treated diabetic rats presented a reduced pancreas Bax/Bcl2 ratio, suggestive of an antiapoptotic effect; in addition, sitagliptin was able to completely reduce (p < 0.001) the pancreas overexpression of IL-1β and TRIB3 found in the untreated diabetic animals; and promoted a significant (p < 0.001) overexpression of VEGF and PCNA.

Conclusion: In this animal model of obese T2DM (the ZDF rat), sitagliptin prevented β-cell dysfunction and evolution of pancreatic damage. The protective effects afforded by this DPP-IV inhibitor may derive from improvement of the metabolic profile (viewed by the amelioration of glucose and TGs levels and of insulin resistance) and from cytoprotective properties, such as antiapoptotic, anti-inflammatory, pro-angiogenic and pro-proliferative.

Figure 1

Sitagliptin effects on inflammation and fibrosis of endocrine pancreas of diabetic ZDF rats. Endocrine inflammation in 26 week-old animals (images A-C): (A) Micrograph of normal islet of Langerhans observed in a lean control ZDF rat; (B) Islet of Langerhans of an untreated diabetic ZDF rat showing severe inflammatory infiltrate occupying over 50% of the islet’s area (Grade 3 inflammation); (C) A clearly regression of inflammation is observed after 6 weeks of sitagliptin treatment in obese diabetic ZDF rat (Grade 1 inflammation). Endocrine fibrosis observed in 26 week-old animals (images D-F): (D) Normal islet of Langerhans of lean control ZDF rat; (E) Grade 3 fibrosis in an untreated obese diabetic ZDF rat, displaying a large and irregularly shaped islet filled with fibrous tissue, evidenced by intense pink staining; (F) Grade 1 fibrosis in a regular, small islet in sitagliptin treated diabetic ZDF rats. Semiquantitative evaluation of inflammation (graph G) and fibrosis (graph H): (G) A significantly higher (p < 0.001) endocrine inflammation in diabetic rats was recorded when compared to lean control animals. Sitagliptin treatment of diabetic ZDF rats during 6 weeks significantly reduced (p < 0.001) inflammation in comparison to untreated counterparts; (H) Data for endocrine fibrosis showed a trend for an increase in diabetic rats when compared to lean control animals, and a trend for improvement with sitagliptin treatment, although without statistical significance. Chi-square test with Monte Carlo simulation or exact test (when contingency tables are 2 × 2) was performed to find out the differences in histomorphological lesions observed in endocrine pancreas. (p < 0.05, p < 0.01 and p < 0.001 for one, two or three symbols, respectively; n = 5 per group).

Figure 2

Sitagliptin effects on inflammation and fibrosis of exocrine pancreas of diabetic ZDF rats. Histopathological observation of exocrine inflammation in 26 week-old animals (images A-C): (A) Micrograph of a normal exocrine parenchyma of lean control ZDF rat; (B) The untreated diabetic rats exhibiting severe inflammatory infiltrate, amid the acini of the exocrine parenchyma (grade 3 inflammatory lesions); (C) Normal exocrine parenchyma with no inflammatory signs in the exocrine pancreas of sitagliptin-treated diabetic ZDF rats. Histopathological observation of exocrine fibrosis in 26 week-old animals (images D-F): (D) Normal pancreatic duct in a lean control ZDF rat; (E) An extremely thickened, grade 3, fibrotic duct, which overextends the microscopic field (interrupted line), with numerous neocanaliculi in the duct wall, .present in an untreated diabetic ZDF rat; (F) Improvement of duct fibrosis from grade 3 to grade 1 (full line) in a 6 weeks sitagliptin treated diabetic ZDF rat. Semiquantitative evaluation of inflammation (graph G) and fibrosis (graph H): (G) Exocrine pancreatic inflammation revealed only a slight descent in sitagliptin treated animals in relation to its untreated counterparts; (H) Sitagliptin presented a trend to decrease the duct fibrosis increment (p < 0.05) found in the untreated ZDF rats. Chi-square test with Monte Carlo simulation or exact test (when contingency tables are 2×2) was performed to find out the differences in histomorphological lesions observed in exocrine pancreas (p < 0.05, p < 0.01 and p < 0.001 for one, two or three symbols, respectively; n = 5 per group).

Figure 3

Sitagliptin protects the diabetic ZDF rats against endocrine pancreas apoptotic cell death. Upper panel (A-C) - pancreas mRNA expression of Bax and bcl2 in 26 week-old ZDF rats: A significant increase (p < 0.05) in apoptotic protein Bax (A), as well as, in antiapoptotic Bcl2 (B) was observed in the untreated diabetic ZDF rats when compared with the lean control ZDF animals, resulting in an unchanged Bax/Bcl2 ratio (C). In the sitagliptin-treated diabetic rats an overexpression of the mRNA was registered for both Bax (not statically significant) and Bcl2 (p < 0.001) ensuing a reduced Bax/Bcl2 ratio (C). Middle panel (D-I) - Immunostaining of pancreas Bax and Bcl2 in 26 week-old ZDF rats: (D) The expression of Bax protein is not present in the pancreatic islet (grade 0) of a lean control rat; (E) A deeply stained islet (grade 3) revealing Bax protein expression in untreated diabetic ZDF rat; (F) The diabetic sitagliptin-treated rats displayed a light stained islet (grade 1); (G) Expression of Bcl2 protein not observed in islet (grade 0) of a lean control rat; (H) A moderately stained islet (grade 2) with Bcl2 antibody in an untreated diabetic ZDF rat; (I) An intensely stained islet (grade 3) in a diabetic sitagliptin-treated rat. Lower panel (J-L) - Quantification of protein expression: Bax protein expression in diabetic untreated rats exhibited a significant increase in relation to lean control rats; in sitagliptin-treated diabetic rats, Bax presented a trend for overexpression (J), accompanied by a significantly (p < 0.001) increased expression of Bcl2 (K), resulting in a Bax/Bcl2 ratio identical to control animals (L). Statistical comparisons between groups were performed using one-way ANOVA and the post-hoc Bonferroni test (p < 0.05, p < 0.01 and p < 0.001 for one, two or three symbols, respectively; n = 5 per group).

Figure 4

Effects of sitagliptin treatment on pancreatic mRNA expression of mediators of inflammation, angiogenesis and proliferation: IL-1β, VEGF and PCNA. (A) mRNA expression of pancreatic IL-1β in untreated diabetic ZDF rats was significantly increased (p < 0.01) in contrast to lean control rats; in the sitagliptin-treated diabetic group, overexpression of IL-1β was significantly prevented (p < 0.001). Overexpression (p < 0.001) of VEGF (B) and PCNA (C) mRNA in ZDF diabetic rats treated with sitagliptin for 6 weeks when compared with untreated diabetic rats. Statistical comparisons between groups were performed using one-way ANOVA and the post-hoc Bonferroni test (p < 0.05, p < 0.01 and p < 0.001 for one, two or three symbols, respectively; n = 5 per group).

Figure 5

Sitagliptin prevents TRIB3 protein overexpression in pancreas of ZDF diabetic rats. Micrographs of TRIB3 expression by immunostaining in 26 week-old ZDF rats (A-C): (A) Lean ZDF control rat presenting unstained pancreatic islet (grade 0); (B) Untreated diabetic ZDF rat showing an intensely stained pancreatic islet (grade 3); (C) Sitagliptin-treated diabetic rat for 6 weeks displayed light staining of endocrine cells (grade 1). Exocrine pancreatic tissue presents normal constant staining for TRIB3 in all three groups. (D) Quantification of protein expression by score points: expression of TRIB3 is almost undetectable in control rats, rising very significantly (p < 0.001) in untreated diabetic ZDF rats; and declining very meaningfully (p < 0.001) in 6 weeks sitagliptin-treated diabetic treated rats. Statistical comparisons between groups were performed using one-way ANOVA and the post-hoc Bonferroni test (p < 0.05, p < 0.01 and p < 0.001 for one, two or three symbols, respectively; n = 5 per group).