Effects of Sitagliptin Treatment on Dysmetabolism, Inflammation, and Oxidative Stress in an Animal Model of Type 2 Diabetes (ZDF Rat

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Effects of Sitagliptin Treatment on Dysmetabolism, Inflammation, and Oxidative Stress in an Animal Model of Type 2 Diabetes (ZDF Rat
  Hindawi Publishing CorporationMediators of InflammationVolume 2010, Article ID 592760, 11 pagesdoi:10.1155/2010/592760 Research Article EffectsofSitagliptinTreatmentonDysmetabolism,Inflammation,andOxidativeStressinanAnimalModelof Type2Diabetes(ZDFRat) LilianaFerreira, 1 EditeTeixeira-de-Lemos, 1,2 FilipaPinto, 1 BelmiroParada, 1 Cristina Mega, 2 Helena Vala, 2 Rui Pinto, 3 Patr´ıcia Garrido, 1 Jos´e Sereno, 1 Rosa Fernandes, 1 PauloSantos, 4 IsabelVelada, 4  AndreiaMelo, 1 SaraNunes, 1 FredericoTeixeira, 1,5 andFl´avioReis 1,5 1 InstituteofPharmacology&ExperimentalTherapeutics,IBILI,MedicineFaculty,UniversityofCoimbra,3000-354Coimbra,Portugal   2 ESAV, Polytechnic Institute of Viseu, 3500 Viseu, Portugal  3 Pharmacology & Pharmacotoxicology Unit, Faculty of Pharmacy, Lisbon University, 1649-003 Lisboa, Portugal  4 Functional Genomics Laboratory, Center of Histocompatibility of the Centre, 3001-301 Coimbra, Portugal  5 Institute for Molecular and Cellular Biology, Porto University, 4150 Porto, Portugal  Correspondence should be addressed to Fl´avio Reis, freis@fmed.uc.ptReceived 28 January 2010; Revised 17 April 2010; Accepted 28 April 2010Academic Editor: Gema Fr¨uhbeck Copyright © 2010 Liliana Ferreira et al.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.The purpose of this paper is to evaluate the chronic e ff  ect of sitagliptin on metabolic profile, inflammation, and redox status inthe Zucker Diabetic Fatty (ZDF) rat, an animal model of obese type 2 diabetes. Diabetic and obese ZDF (  fa/fa ) rats and theircontrols (ZDF +/+) were treated during 6 weeks with vehicle (control) and sitagliptin (10mg/kg/bw). Glucose, HbA1c, insulin,Total-c, TGs, IL-1  β , TNF- α , CRP hs , and adiponectin were assessed in serum and MDA and TAS in serum, pancreas, and heart.Pancreatic histology was also evaluated. Sitagliptin in diabetic rats promoted a decrease in glucose, HbA1c, Total-c, and TGsaccompanied by a partial prevention of insulinopenia, together, with a decrease in CRP hs  and IL-1  β . Sitagliptin also showed apositive impact on lipid peroxidation and hypertension prevention. In conclusion, chronic sitagliptin treatment corrected theglycaemic dysmetabolism, hypertriglyceridaemia, inflammation, and hypertension, reduced the severity of the histopathologicallesions of pancreatic endocrine and exocrine tissues, together with a favourable redox status, which might be a further advantagein the management of diabetes and its proatherogenic comorbidities. 1.Introduction Type 2 diabetes mellitus (T2DM) is the most commonendocrine disorder worldwide, a ff  ecting more than 200million people [1]. Pathogenesis of this disease involvesabnormalities in glucose and lipid metabolism, includinginadequate insulin secretion from pancreatic  β -cells andresistance to insulin activity (insulin resistance) [2].Hyperglycaemia and hyperlipidaemia are the key pro-moters, through distinct mechanisms, of reactive oxygenspecies (ROS) and advanced glycation end products (AGEs)production, which causes cell damage and insulin resistance[3, 4]. Moreover, these high levels of glucose and lipids stimulate pro-inflammatory cytokines, promote lipid perox-idation, thus contributing to beta-cell degradation, particu-larly due to apoptosis pathways [5]. Actually, inflammationand oxidative stress play a major role in type 2 diabetesmellitus (T2DM) pathophysiology, contributing for obesity,insulin resistance and cardiovascular complications, whichfurther aggravate the disease. However, so far, there areno therapeutic options able to e ffi ciently act not only onthe glucose control but, and specially, on the prevention of T2DM evolution and its complications, namely, by beta-cellfunction preservation.In T2DM patients, the e ff  ect of the glucose-dependentinsulinotropic polypeptide (GIP), as well as the secretion  2 Mediators of Inflammationof the glucagon-like peptide-1 (GLP-1), is diminished orabsent,contributingtoinsulinsecretiondeficiency[6].Thesetwo incretins are secreted by the intestine [7] and stimu-late insulin secretion by beta-cells, in a glucose-dependentmanner [8], preventing hypoglycemia. In animal models,continuous infusion of GLP-1 or injection of long-actingGLP-1 mimetics, such as exendin-4, has shown a remarkableglucose-lowering e ffi cacy, together with an ability to increasebeta-cell neogenesis and reduce apoptosis and alpha-cellglucagon secretion [9–11]. Despite the beneficial actions of  GLP-1 and GIP, their use as antidiabetic agents (mimetics)is impractical due to their short half-lives, as a result of their rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) [12, 13]. Thus, orally administered DPP-IV inhibitors have emerged as a new class of antihyperglycaemic agentswith the ability for extending the biological e ff  ects of incretinhormones through the inhibition of their degradation [14,15], with the advantage of higher stability and bioavailability when compared with the mimetics.Sitagliptin, an orally available DPP-IV inhibitor devel-oped to be used as a once daily treatment for T2DM,has shown beneficial e ff  ects on glycaemic control, reducingHbA1c, and preventing hypoglycemia, as well as on isletmass and function, with no relevant adverse e ff  ects [16,17]. Considering the vast physiological actions promotedby the incretins, not only related with the control of glucose by insulin and glucagon regulation, but also withthe peripheral insulin sensitization, cardiac and neuronalprotection and beta-cell preservation, the use of an incretinenhancer (such as sitagliptin) might present beneficial e ff  ectson diabetes pathophysiology and on prevention of its seriouscomplications, which deserves better elucidation.The male Zucker Diabetic Fatty (ZDF) rat displaysglucose intolerance, marked insulin resistance, and hyper-lipidaemia, and becomes overtly diabetic after 8 weeks of age if fed a diet containing 6.5% fat [18]. In the prediabeticstate, the male ZDF rat experiences a steady increase inbasal insulinaemia and plasma free fatty acid (FFA) levels.Hyperglycemia develops between 8 and 10 weeks of age,leading to overt diabetes and collapsing insulin secretion[19]. This profile mimics the progressive loss of glucose-stimulated insulin secretion in human type 2 diabetes and,thus, the ZDF rat represents a good animal model forstudying human T2DM pathophysiology and the e ff  ects of therapeutic options [20].The purpose of this study was, thus, to assess the e ff  ectsof chronic sitagliptin treatment on the metabolic profile,inflammation, and redox status and pancreashistology in theZDF rat, an animal model of obese T2DM. 2.MaterialandMethods  2.1. Animals and Experimental Design.  Male ZDF rats(ZDF/Gmi,  fa/fa ) and their littermates (ZDF/Gmi, +  /  +)were purchased from Charles River Laboratories (Barcelona,Spain) with 6 weeks of age. Rats were properly housed,handled daily, and kept at a controlled standard temperature(22-23 ◦ C), humidity (60%) and light-dark cycles (12/12hours). Throughout the experiment, the animals were feddistilled water  ad libitum  and rodent maintenance chow (A-04 Panlab, Barcelona, Spain) containing 15.4% of proteinand 2.9% of lipids). The chow was adapted to the animal’sbody weight (BW): 100mg/g. Animal experiments wereconducted according to the European Council Directives onAnimal Care and to the National Laws.When aged 20 weeks (T0), the diabetic ZDF (  fa/fa )rats were divided in 2 subgroups ( n  =  8 rats each): acontrolandatreatmentgroup,receiving,respectively,byoralgavage, once a day (6:00 PM), during 6 weeks, the vehicle(orange juice) and sitagliptin (10mg/kg/BW/day). The sameprocedures were adopted with the lean nondiabetic ZDF(+  /  +) control rats. The ZDF (+  /  +) control group undersitagliptin treatment showed no relevant di ff  erences whencompared with the ZDF (+  /  +) control rats under vehicleand, thus, the results were excluded from tables and figuresin order to facilitate data comparison and interpretation.Food intake and BW were measured each day beforetreatment and expressed as weekly average values. Systolicblood pressure (SBP), diastolic blood pressure (DBP) andheart rate (HR) were determined in conscious rats usinga tail-cu ff   sphygmomanometer LE 5001 (Letica, Barcelona,Spain) in appropriate restriction cages. Pulse pressure (PP)was calculated by the di ff  erence between the systolic andthe diastolic readings (PP = SBP − DBP). Blood pressure (BP)values, obtained by averaging 8 to 10 measurements, wererecorded by the same person, in a similar peaceful environ-ment. Measurements were performed at T0 and at the end of the study (Tf) with special precautions to minimize stress-induced fluctuations in BP, as previously described [21].  2.2. Sample Collection and Preparation. Blood  : when aged 20weeks (T0) and at the end of the experience (26 weeks -Tf) the rats were subjected to intraperitoneal anesthesia witha 2mg/kg BW of a 2:1 (v:v) 50mg/mL ketamine (Ketalar,Parke-Davis, Lab. Pfeizer Lda, Seixal, Portugal) solution in2.5% chlorpromazine (Largactil, Rhˆone-Poulenc Rorer, Lab.Vit´oria, Amadora, Portugal) and blood samples were imme-diately collected by venipuncture from the jugular vein intosyringes without anticoagulant (for serum samples) or withthe appropriate anticoagulant: ethylene-diaminetetraaceticacid (EDTA)-2K for Glycosylated haemoglobin (HbA 1 c)measurement.The rats were sacrificed by anesthetic overdose. Thepancreas and the heart were immediately removed, placedin ice-cold Krebs’ bu ff  er and Bock’s fixative, respectively,and carefully cleaned of extraneous fat, lymph nodes andconnective tissue. The organs were cross-sectioned andcryopreservated, fixed and processed for para ffi n embeddingin accordance with subsequent analysis protocols.  2.3. Glycaemic and Lipidic Profile Assays.  Serum total choles-terol (Total-c) and triglycerides (TGs) were analysed on aHitachi 717 analyser (Roche Diagnostics) using standardlaboratorial methods. Total-c reagents and TGs kit wereobtained from bioM´erieux (Lyon, France). Serum glucoselevels were measured using a Glucose oxidase commercialkit (Sigma, St. Louis, Mo, USA). Considering the variability of serum glucose levels in the rat, glycosylated haemoglobin  Mediators of Inflammation 3 Table  1: Body weight, lipid profile and blood pressure in the control and diabetic ZDF rats at the initial and final time (6 weeks of vehicle orsitagliptin treatment).Initial Time (20 wks) Final Time (26 wks)Control ZDF (+  /  +) Diabetic ZDF (  fa/fa ) Control ZDF (+/+) Diabetic ZDF (  fa/fa )Groups ( n = 16) ( n = 16) Vehicle ( n = 8) Vehicle ( n = 8) Sitagliptin ( n = 8)BW (g) 406 . 70 ± 6 . 83 388 . 10 ± 8 . 87 445 . 70 ± 8 . 16 354 . 40 ± 8 . 85 aaa 380.00 ± 14.46Total-c (mg/dl) 77 . 50 ± 1 . 50 155 . 50 ± 3 . 50 aaa 93 . 00 ± 2 . 96 193 . 00 ± 9 . 79 aaa 193.10 ± 4.62TGs (mg/dl) 115 . 00 ± 11 . 00 374 . 50 ± 4 . 95 a 154 . 00 ± 19 . 14 400 . 20 ± 27 . 00 aaa 237 . 10 ± 22 . 54 bbb Systolic (mmHg) 115 . 50 ± 0 . 83 125 . 20 ± 0 . 27 116 . 00 ± 2 . 52 127 . 80 ± 1 . 23 a 101 . 60 ± 0 . 78 bbb Diastolic (mmHg) 100 . 98 ± 0 . 82 91 . 46 ± 0 . 83 103 . 50 ± 1 . 94 112 . 70 ± 3 . 98 94 . 86 ± 0 . 70 bbb Mean (mmHg) 104 . 25 ± 0 . 25 108 . 20 ± 1 . 42 104 . 30 ± 4 . 25 117 . 40 ± 3 . 04 a 96 . 86 ± 0 . 51 bbb Pulse P (mmHg) 14 . 52 ± 0 . 98 33 . 74 ± 0 . 37 14 . 00 ± 4 . 16 15 . 09 ± 3 . 08 6 . 71 ± 1 . 11 b BW, body weight; P, pressure; SITA, sitagliptin; Total-c, Total-cholesterol; TGs, triglycerides; ZDF, Zucker diabetic fatty. Values are means ± SEM of   n  rats.Comparisons between groups: a - ZDF (  fa/fa ) versus ZDF (+  /  +) and b - sitagliptin versus vehicle;  P < . 05,  P < . 01 and  P < . 001 for one, two or three letters,respectively. (HbA 1 c) levels were used as an index of glucose control,throughtheDCA2000+ lateximmunoagglutination method(Bayer Diagnostics, Barcelona, Spain). Plasma insulin levelswere quantified by using a rat insulin Elisa assay kit fromMercodia(Uppsala,Sweden).Insulinsensitivityofindividualanimals was evaluated using the previously validated home-ostasis model assessment (HOMA) index [21]. The formulaused was as follows: [HOMA-IR]  =  fasting serum glucose(mmol/l) x fasting serum insulin ( µ U/ml)/22.5. The valuesused (insulin and glucose) were obtained after an overnightof food deprivation.  2.4. Inflammatory Profile and Redox Status.  Serum levels of interleukin-1  β  (IL-1  β ), tumour necrosis factor  α  (TNF- α )and adiponectin were all measured by rat-specific Quan-tikine ELISA kits from R&D Systems (Minneapolis, USA).High-sensitive C-reactive protein (CRP hs ) was determinedby using a rat-specific Elisa kit from Helica Biosystems Inc.(Fullerton, CA, USA). All assays were performed accordingto the manufacturers’ recommendations, in duplicate.The thiobarbituric acid reactive-species (TBARs) assay was used to assess serum, pancreas and heart products of lipid peroxidation, via malondialdehyde (MDA), accord-ing to that previously described in [22]. Samples wereanalysed spectrophotometrically at 532nm using 1,1,3,3-tetramethoxypropane as external standard. The concentra-tion of lipid peroxides (in MDA) was expressed as  µ mol/l inthe plasma and as  µ mol/g tissue in the pancreas and heart.Ferric reducing antioxidant potential (FRAP) assay was usedto estimate serum total antioxidant status (TAS) [23].  2.5. Histological Studies.  Specimens were para ffi n-embeddedand the 3 µ m thick sections stained for routine histopatho-logical diagnosis with haematoxylin and eosin (HE). Allsamples were examined by light microscopy using a Micro-scope Zeiss Mod. Axioplan 2. The degree of injury visibleby light microscopy was scored in a single-blind fashionby the pathologist to the animal study group. Endocrinepancreatic damage was assessed by evaluating changes inthe islets of Langerhans, namely the shape (architecture),presence of inflammatory infiltrate, fibrosis, vacuolizationand intraislets congestion. A semiquantitative rating foreach slide ranging from 0 (minimal) to 3 (severe andextensive damage) was assigned to each component. Theexocrine pancreatic damage was evaluated, according to thepresence of congestion, fibrosis, and inflammatory infiltratein the interstitial tissues and graded, also, in the samesemiquantitative rating.  2.6. Statistical Analysis.  Results are shown as mean  ± standard error of the mean (SEM). The comparison of valuesbetweengroupswasperformedbyusingANOVAfollowedby the Bonferroni post hoc test, through appropriate software(GraphPadPrism 5.0 from GraphPad Software Inc., La Jolla,CA, USA). Significance was accepted at a  P   less than  . 05. 3.Results 3.1. E   ff  ects of Chronic Sitagliptin Treatment on Body Weight and Glycaemic and Lipidic Profiles.  Concerning the body weight, no significant di ff  erences were encountered betweenthe diabetic and the lean control rats in the beginningof treatments (T0: week 20), despite the obese profileencountered in the diabetic ZDF (  fa/fa ) rats between the 8thand the 14th week (data not shown). At the end of the study (26 weeks), the control diabetic ZDF (  fa/fa ) rats exhibit an8.7% reduction in their BW ( P < . 001); nevertheless, the leancontrol group gained weight. Sitagliptin treatment, during 6weeks,stabilized theloss ofweightin thediabetic ZDF  (fa/fa) rats, even preventing part of the BW loss when comparedwith the rats without treatment (Table 1).The determination of serum glucose, HbA1c, Total-c andTGs concentrations was carried at the initial time (T0: 20weeks old) and at the end of the study (Tf: 26 weeks old).At the T0, the diabetic group showed a hyperglycaemic and ahyperlipidemicprofile,alsoseenatthefinaltime(Figure 1(a)and Table 1). As illustrated in Figure 1(b), the HbA1c values were higher in the diabetic rats than those of the controlanimals,confirmingtheglycaemicderegulation.ThediabeticZDF (  fa/fa ) rats have also presented higher levels of Total-c  4 Mediators of Inflammation 0510152025Initial T Final Taaaaaa bbb     G     l   y   c   a   e   m    i   a     (   m   m   o     l    /    L     ) (a)00 . 050 . 10 . 15Initial T Final Taaa aaa bbb     H     b    A    1   c     (   p   r   o   p   o   r    t    i   o   n   o     f    t   o    t   a     l    H     b     ) (b)00 . 511 . 522 . 5Initial T Final Tb     I   n   s   u     l    i   n     (      µ    g    /     l     ) Control ZDF (+/+)Diabetic ZDF (  fa/fa )Diabetic ZDF (  fa/fa ) + SITA(c)0510152025Initial T Final Taaa aaa bbb     H    O    M    A  -    I    R Control ZDF (+/+)Diabetic ZDF (  fa/fa )Diabetic ZDF (  fa/fa ) + SITA(d) Figure  1: Glycaemic and insulinaemic profiles .  Serum Glycaemia (a), HbA1c (b), insulinaemia (c) and insulin resistance (HOMA-IR) index (d), for the control (+  /  +) and diabetic (  fa/fa ) ZDF rats, in the initial and final times (6 weeks of vehicle or 10mg/kg BW/day sitagliptintreatment). Comparisons between groups ( n  =  8 each): a - ZDF (  fa/fa ) versus ZDF (+  /  +) and b - with sita versus without sita;  P < . 05, P < . 01 and  P < . 001 for one, two or three letters, respectively. HOMA-IR, homeostasis model assessment—insulin resistance. and TGs versus the control ZDF (+  /  +) animals, in both times(Table 1).After 6 weeks of sitagliptin treatment (Tf: 26 weeks), asignificant improvement in glycemic control was observedin diabetic ZDF (  fa/fa ) rats (486.3  ±  19.1mg/dl), whencompared with the vehicle-treated diabetic animals (523.3 ± 15.6mg/dl;  P < . 001) (Figure 1(a)). This pattern of changesis also expressed by the HbA1c levels, which decreased by 11.1% in sitagliptin-treated ZDF (  fa/fa ) rats when comparedwith the diabetic rats not treated with the drug (Figure 1(b)).TGs were significantly reduced (50%;  P < . 001) in thediabetic rats treated with sitagliptin during 6 weeks versusthe diabetic vehicle-treated group (Table 1). 3.2. E   ff  ects of Chronic Sitagliptin Treatment on Insulin Levelsand Insulin Resistance (HOMA-IR).  At the beginning of the study (T0), insulin levels were higher in the diabeticrats than those of the control, but the di ff  erences did notreach statistical significance. At the final time, the vehicle-treated ZDF (  fa/fa ) rats exhibit relative insulinopenia (0 . 75 ± 0 . 05 µ g  /  l), when compared to vehicle-treated ZDF (+  /  +)(1 . 05 ± 0 . 30 µ g  /  l) (Figure 1(c)), accompanied by a significantaugment ( P < . 001) of insulin resistance (HOMA-IR index) (Figure 1(d)). The elevation of insulin resistance wasprevented( P < . 001)inthesitagliptin-treateddiabetic(  fa/fa )rats (Figure 1(d)). 3.3. E   ff  ects of Chronic Sitagliptin Treatment on Blood Pressure. The vehicle-treated ZDF (  fa/fa ) group showed significantly ( P < . 05) higher levels of systolic and mean BP, together witha trend to higher diastolic and pulse pressure, when com-pared with the vehicle-treated ZDF (+  /  +) group. Sitagliptin  Mediators of Inflammation 5 0102030Initial T Final Tbbb     C    R    P     h   s     (   m   g    /   m    L     ) (a)050100150Initial T Final Taa a b     I    L  -    1        β      (   p   g    /   m    L     ) (b)010203040Initial T Final Ta bb     T    N    F  -      α      (   p   g    /   m    L     ) Control ZDF (+/+)Diabetic ZDF (  fa/fa )Diabetic ZDF (  fa/fa ) + SITA(c)01234Initial T Final Taa     A     d    i   p   o   n   e   c    t    i   n     (   m   g    /   m    L     ) Control ZDF (+/+)Diabetic ZDF (  fa/fa )Diabetic ZDF (  fa/fa ) + SITA(d) Figure  2: Serum inflammatory markers. Serum CRP hs  (a), IL-1  β  (b), TNF- α  (c) and Adiponectin (d) for the control (+  /  +) and diabetic(  fa/fa ) ZDF rats, in the initial and final times (6 weeks of vehicle or 10mg/kg BW/day sitagliptin treatment). Comparisons between groups( n  =  8 each): a - ZDF (  fa/fa ) versus ZDF (+  /  +) and b - with sita versus without sita;  P < . 05,  P < . 01 and  P < . 001 for one, two or threeletters, respectively. CRP hs , high-sensitive C-reactive protein; IL-1  β , interleukin-1beta; TNF- α , Tumor necrosis factor-alpha. treatment has significantly prevented the blood pressure rise(hypertension) in the diabetic rats (Table 1). 3.4. E   ff  ects of Chronic Sitagliptin Treatment on Inflammatory Profile.  Concerning the serum CRP hs  levels, no significantdi ff  erences were observed between the diabetic ZDF (  fa/fa )and the nondiabetic ZDF (+  /  +) vehicle-treated groups(Figure 2(a)). However, there was higher serum levels of IL-1  β  and TNF- α  and reduced of adiponectin in the vehicle-treated diabetic ZDF (  fa/fa ) rats when compared with thevehicle-treatednondiabetic(+  /  +)rats(Figures2(b),2(c)and 2(d)). Sitagliptin treatment has significantly decreased thelevels of CRP hs  ( P < . 001) and IL-1  β  ( P < . 05) in the diabeticZDF rats (Figures 2(a) and 2(b)). However, the diabetic (  fa/fa ) animals under stagliptin therapy showed, at the endof the study, elevated ( P < . 01) levels of TNF- α  (Figure 2(c)),without significant changes on serum adiponectin contents(Figure 2(d)). 3.5. E   ff  ects of Chronic Sitagliptin Treatment on Serum and Tissue Redox Status.  The vehicle-treated diabetic ZDF (  fa/fa )group exhibited significantly higher levels of serum MDA (atthe T0 and Tf), accompanied by a compensatory elevationof TAS in the final time (Figures 3(a) and 3(b)). Sitagliptin treatment during 6 weeks has decreased ( P < . 01) serum TAScontent, whereas there were no di ff  erences in serum MDAlevels (Figures 3(a) and 3(b)). On the contrary, we observed a significant reduction of pancreas ( P < . 001) and heart( P < . 001) MDA levels in the sitagliptin-treated diabetic ZDF(  fa/fa ) rats when compared with the vehicle-treated (  fa/fa )rats (Figures 3(c) and 3(d)). 3.6. E   ff  ects of Chronic Sitagliptin Treatment on Pancreatic His-tology.  In the control rats (ZDF (+  /  +) under vehicle treat-ment, there was no pathological changes in the endocrineand exocrine pancreas (Figure 4(a)). Langerhans islets of diabetic ZDF animals treated with sitagliptin presented a
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