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  ORIGINAL INVESTIGATION Open Access Early cardiac changes in a rat model of prediabetes: brain natriuretic peptideoverexpression seems to be the best marker Sara Nunes 1 , Edna Soares 1 , João Fernandes 2,3 , Sofia Viana 1 , Eugénia Carvalho 4,5 , Frederico C Pereira 1 and Flávio Reis 1* Abstract Background:  Diabetic cardiomyopathy (DCM) is defined as structural and functional changes in the myocardiumdue to metabolic and cellular abnormalities induced by diabetes mellitus (DM). The impact of prediabeticconditions on the cardiac tissue remains to be elucidated. The goal of this study was to elucidate whether cardiacdysfunction is already present in a state of prediabetes, in the presence of insulin resistance, and to unravel theunderlying mechanisms, in a rat model without obesity and hypertension as confounding factors. Methods:  Two groups of 16-week-old Wistar rats were tested during a 9 week protocol: high sucrose (HSu) dietgroup (n = 7)  –  rats receiving 35% of sucrose in drinking water vs the vehicle control group (n = 7). The animalmodel was characterized in terms of body weight (BW) and the glycemic, insulinemic and lipidic profiles. Thefollowing parameters were assessed to evaluate possible early cardiac alterations and underlying mechanisms:blood pressure, heart rate, heart and left ventricle (LV) trophism indexes, as well as the serum and tissue proteinand/or the mRNA expression of markers for fibrosis, hypertrophy, proliferation, apoptosis, angiogenesis, endothelialfunction, inflammation and oxidative stress. Results:  The HSu-treated rats presented normal fasting plasma glucose (FPG) but impaired glucose tolerance (IGT),accompanied by hyperinsulinemia and insulin resistance (P < 0.01), confirming this rat model as prediabetic.Furthermore, although hypertriglyceridemia (P < 0.05) was observed, obesity and hypertension were absent.Regarding the impact of the HSu diet on the cardiac tissue, our results indicated that 9 weeks of treatment mightbe associated with initial cardiac changes, as suggested by the increased LV weight/BW ratio (P < 0.01) and aremarkable brain natriuretic peptide (BNP) mRNA overexpression (P < 0.01), together with a marked trend for anupregulation of other important mediators of fibrosis, hypertrophy, angiogenesis and endothelial lesions, as well asoxidative stress. The inflammatory and apoptotic markers measured were unchanged. Conclusions:  This animal model of prediabetes/insulin resistance could be an important tool to evaluate the earlycardiac impact of dysmetabolism (hyperinsulinemia and impaired glucose tolerance with fasting normoglycemia),without confounding factors such as obesity and hypertension. Left ventricle hypertrophy is already present andbrain natriuretic peptide seems to be the best early marker for this condition. Keywords:  Brain natriuretic peptide, Diabetic cardiomyopathy, Fibrosis, Hypertrophy, High-sucrose diet, Prediabetes * Correspondence: freis@fmed.uc.pt 1 Laboratory of Pharmacology and Experimental Therapeutics, IBILI, Faculty of Medicine, University of Coimbra, Coimbra 3000-548, PortugalFull list of author information is available at the end of the article C ARDIO V ASCULAR D IABETOLOGY © 2013 Nunes et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the srcinal work is properly cited. Nunes  et al. Cardiovascular Diabetology   2013,  12 :44http://www.cardiab.com/content/12/1/44  Background Diabetic cardiomyopathy (DCM) is defined as structuraland functional changes in the myocardium, independentof hypertension, chronic artery disease (CAD) or any other known cardiac diseases, which are caused by metabolic and cellular abnormalities induced by diabetesmellitus (DM), ultimately resulting in heart failure (HF)[1-3]. Cardiovascular diseases (CVD) are responsible for three quarters of the deaths among the diabetic popula-tion [4] and diabetes accounts for a significant percent-age of patients with a diagnosis of HF, which is a majorcause of mortality and morbidity in these patients [5,6]. The transition from the early metabolic abnormalitiesthat precedes diabetes, for example impaired fasting glu-cose (IFG) and impaired glucose tolerance (IGT), to dia-betes may take many years; however, current estimatesindicate that most individuals with these pre-diabeticstates eventually develop DM [7-9]. DCM may be sub- clinical for a long time, before the appearance of clinicalsymptoms or signs, complicating its diagnosis and aggra- vating future complications. However, during the predia-betic state, the risk of cardiovascular events is already increased and myocardial abnormalities might appearprior to the diagnosis of type 2 DM. Thus, the earlieridentification of cardiac changes in prediabetic/insulinresistant patients could be a better strategy to preventthe evolution to most serious stages of the disease.One of the most important structural hallmarks of DCM is cardiac hypertrophy [10,11], and this, in turn, is a powerful predictor of CV events [12]. Hyperglycemiahas been viewed as the pivotal pathogenetic factor forthe development of DCM. In fact, it can cause abnor-malities at the cardiac myocyte level, eventually leadingto functional and structural abnormalities, including sys-tolic and diastolic dysfunction, as well as cardiac hyper-trophy and myocardial fibrosis [13]. However, otherfactors seem to be involved in the evolution of the dis-ease, including hyperinsulinemia, insulin resistance, oxi-dative stress, inflammation, endothelial dysfunction andapoptosis [10,11,14]. During the last years, the patho- logical, structural, functional and molecular aspects of the disease have been increasingly investigated, but theissue is far to being elucidated. The knowledge of thecellular and molecular aspects underlying the metabolicdisturbances on cardiomyocytes in the prediabetic stateshould be useful in predicting the structural and func-tional cardiac consequences. Animal models have beenused to study the mechanisms underlying DCM [15-18], but there are limitations. Indeed, the presence of import-ant confounding factors displaying the susceptibility tocardiomyopathy, such as obesity and hypertension, aswell as the scarce animal models of prediabetes aremajor impediments in the progress and understandingof the disease.Thus, the purpose of this work was to elucidatewhether cardiac alteration are already present in a prediabetic state, and to study its underlying mechanisms,using the high-sucrose (HSu) diet rat model, which is as-sociated with minor metabolic abnormalities and mightmimic the human prediabetic state of insulin resistance[19,20], without other complicating factors that could lead to cardiac events. Material and methods Animals and diet Male adult (16 weeks-old) Wistar rats (Charles River La-boratories, Barcelona, Spain), weighting 332.9 ±9.0 gwere maintained under controlled temperature (22 – 23°C)and light (12:12-h light – dark cycle). After 1 week of acclimatization, animals were randomly divided into twogroups (n=7 each), for a 9-weeks protocol: control  –  ratscontinued to receive tap water for drinking; and high-sucrose diet (HSu), rats received 35% sucrose (S0389;Sigma-Aldrich) in the drinking water. All animals werefed standard rat chow, containing 16.1% of protein, 3.1%of lipids, 3.9% of fibers and 5.1% of minerals (AO4 Panlab,Barcelona, Spain)  ad libitum  (with exception in the fastingperiods). Food and beverage consumption was monitoredfor both groups throughout the experiment. All experi-ments were conducted according to the National andEuropean Directives on Animal Care, as well as with AR-RIVE guidelines on animal research [21]. The body weight(BW) of each animal was recorded weekly during the ex-perimental period, using an analytical balance (KERN CB6 K1, Germany). Blood pressure and heart rate assessment Systolic (SBP), diastolic (DBP), mean blood pressure(MBP) and heart rate (HR) values were evaluated inconscious rats using a tail-cuff sphygmomanometer LE5001 (Letica, Barcelona, Spain) in appropriate conten-tion cages. Blood and tissue collection and preparation At the final time, the rats were subjected to intraperitonealanesthesia with 50 mg/kg pentobarbital (Sigma-Aldrich,Portugal) solution and a blood sample was immediately collected by venipuncture from the jugular vein intosyringes with Heparin-Lithium (Sarstedt, Monovette W ) forplasma samples and into needles without anticoagulant(for serum samples). The rats were then sacrificed by cer- vical dislocation, and the heart was immediately removed,placed in ice-cold Krebs ’  buffer, carefully cleaned of adher-ent fat and connective tissue, weighted and divided in leftand right ventricle. Heart regions were frozen in liquid ni-trogen and stored at  − 80°C, for biochemical or gene ex-pression analysis. Before sampling, heart weight (HW) andleft ventricle weight (LVW) were measured in order to be Nunes  et al. Cardiovascular Diabetology   2013,  12 :44 Page 2 of 11http://www.cardiab.com/content/12/1/44  used as cardiac trophism indexes in relation to body weight: HW/BW and LVW/BW. Metabolic characterization Glucose tolerance test (GTT) was performed in fastedrats (6-h) injected intraperitoneally (i.p.) with a glucosebolus of 2 g/kg BW. The tail vein blood glucose levelswere measured using a portable device (One TouchUltraEasy  W glucometer, Lifescan, Johnson and Johnson,Portugal) in samples taken immediately before the bolusand 15, 30, 60, and 120 minutes after. Glycemia wasmeasured in fed conditions.Insulin tolerance test (ITT) was performed after an i.p. injection of 0.75 U/kg BW of insulin (I9278, Sigma),in 6-h fasted rats, through monitoring the blood glucosebefore the injection and 15, 30, 45, 60 and 120 min.after, using the same glucometer. Fasting insulin levelswere quantified by using a rat insulin ELISA kit(Mercodia, Uppsala, Sweden). Insulin sensitivity of indi- vidual animals was evaluated using the homeostasismodel assessment (HOMA) index [22]. The formulaused was as follows: [HOMA-IR] = fasting serum glu-cose (mg/dL) × fasting serum insulin ( μ U/mL)/22.5. The values used (insulin and glucose) were obtained after anovernight fasting period.Serum total cholesterol (TC) and triglycerides (TGs)were analyzed by enzymatic methods using an auto-matic analyzer (Hitachi 717, Roche Diagnostics). Total-cholesterol reagents and TGs kits were obtained frombioMérieux (Lyon, France). Serum and heart muscle protein levels and redox status Serum levels of transforming growth factor  β -1 (TGF- β 1), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) were measured through micro-ELISA sandwichassay, using commercial ultrasensitive Quantikine W ELISAkits (R&D Systems, Minneapolis, USA).Cardiac expression of the receptor for advancedglycation endproducts (RAGE) and of tumour necrosisfactor alpha (TNF- α ) were quantified by western blot.The extracts were obtained from the left ventricle andwere homogenized in 1.5 ml of RIPA lysis buffer(150 mM NaCl; 50 mM Tris – HCl pH =8.0; 5 mMEGTA; 1% Triton X-100; 0.5% DOC; 0.1% SDS), sup-plemented with a protease inhibitor cocktail (1 mMphenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1  μ g/mL chymostatin, 1  μ g/mL leupeptin, 1  μ g/mL antipainand 5  μ g/mL pepstatin A; Sigma-Aldrich) and centrifuged3 times (15500 × g, 15 min., 4°C). The resulting super-natant fraction (corresponding to total extract) was col-lected and total protein concentration was determinedusing BCA assay [23], supernatants were stored at  – 80°Cuntil further use. Known amounts of total protein (25  μ gfor RAGE and 90  μ g for TNF- α ) were loaded andseparated by electrophoresis on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (7.5% and 10%, res-pectively), transferred to a 0.45  μ m polyvinylidene difluo-ride (PVDF) membranes (Immobilon, Millipore, Madrid,Spain) and blocked with 1% bovine serum albumin (BSA)in phosphate buffer saline with 0.1% Tween-20 (PBS-T)for 1 h at room temperature. Membranes were thenincubated overnight at 4°C with primary antibody (rabbitanti-RAGE, 1:1000; anti-TNF- α , 1:600) both from Abcam(Cambridge, UK). The membranes were washed exten-sively in 0.1% PBS-T and then incubated for 1 h at roomtemperature with alkaline phosphatase conjugated sec-ondary antibodies (goat anti-rabbit, 1:5000, GE Healthcare).Finally, membranes were visualized on Typhoon FLA 900(GE Healthcare Bio-sciences) imaging system, using an en-hanced chemifluorescence detection reagent (ECF, GEHealthcare). To confirm equal protein loading and sampletransfer, membranes were reprobed with  β -actin (1:5,000;Sigma-Aldrich) antibodies. Densitometric analyses wereperformed using the Image Quant 5.0 software. Resultswere normalized against  β -tubulin, and then expressed aspercentage of control.The thiobarbituric acid reactive-species (TBARs) assay was used to evaluate serum and heart muscle tissue lipidperoxidation, via malondialdehyde (MDA). Sampleswere analyzed spectrophotometrically at 532 nm using1,1,3,3-tetramethoxypropane as an external standard.Serum total antioxidant status (TAS) was measuredthrough ferric reducing antioxidant potential (FRAP)assay, as previously described [24]. RT-qPCR cardiac muscle gene expression The heart was isolated and stored in RNA later ™  solution(Ambion, Austin, TX, USA). In brief, tissue lysates wereprocessed according to the protocol from RNeasy  W MiniKit (Qiagen, Hilden, Germany). RNA integrity (RIN,RNA Integrity Number) was analyzed using 6000 NanoChip W kit, in Agilent 2100 bioanalyzer (Agilent Tech-nologies, Walbronn, Germany) and 2100 expert soft-ware, following manufacturer instructions. The yieldfrom isolation was from 0.5 to 3  μ g; RIN values were6.0 – 9.0 and purity (A260/A280) was 1.8 – 2.0.  Reversetranscription and relative quantification of gene expres- sion were  performed as previously described [25]. Real-time PCR reactions were performed using the followingprimer sequences for Bax, Bcl2, brain natriuretic pep-tide (BNP), connective tissue growth factor (CTGF),intercellular adhesion molecule 1 (ICAM-1), IL-6,inducible nitric oxide synthase (iNOS), Pro-collagentype III, superoxide dismutase (SOD), TGF- β 1, TNF- α ,thrombospondin 1 (TSP-1), tribbles 3 (TRB3), vascularcell adhesion molecule 1 (VCAM-1) and VEGF, whichwere normalized in relation to the expression of beta-actinas an internal control: Bax Forward: 5 0 -CCAAGAAGC Nunes  et al. Cardiovascular Diabetology   2013,  12 :44 Page 3 of 11http://www.cardiab.com/content/12/1/44  TGAGCGAGTGTCTC-3 0 and Bax Reverse: 5 0 -AGTTGCCATCAGCAAACATGTCA-3 0 ; Bcl-2 Forward: 5 0 -GGAGCGTCAACAGGGAGATG-3 0 and Bcl-2 Reverse: 5 0 -GATGCCGGTTCAGGTACTCAG-3 0 ; BNP Forward: 5 0 -GGGCTGTGACGGGCTGAGGTT-3 0 and BNP Reverse:5 0 -AGTTTGTGCTGGAAGTAAGA-3 0 ; CTGF Forward:5 0 -CGTAGACGGTAAAGCAATGG-3 0 and CTGF Re- verse: 5 0 -AGTCAAAGAAGCAGCAAACAC-3 0 ; ICAM-1Forward: 5 0 -TTCAACCCGTGCCAGGC-3 0 and ICAM-1Reverse: 5 0 -GTTCGTCTTTCATCCAGTTAGTCT-3 0 ; IL-6Forward: 5 0 -ACCACTTCACAAGTCGGAGG-3 0 and IL-6Reverse: 5 0 -ACAGTGCATCATCGCTGTTC-3 0 ; iNOSForward: 5 0 -CAGAAGCAGAATGTGACCATCAT-3 0 andiNOS Reverse: 5 0 -CGGAGGGACCAGCCAAATC-3 0 ; Pro-collagen type III Forward: 5 0 -5 0 -GGTCACTTTCACTGGTTGACGA-3 0 and Pro-collagen type III Reverse:5 0 -TTGAATATCAAACACGCAAGGC-3 0 ; SOD Forward:5 0 -GACAAACCTGAGCCCTAACGG-3 0 and SOD Re- verse: 5 0 -CTTCTTGCAAACTATG-3 0 ; TGF- β 1 Forward:5 0 -ATACGCCTGAGTGGCTGTCT-3 0 and TGF- β 1 Re- verse: 5 0 -TGGGACTGATCCCATTGATT-3 0 ; TNF- α  For-ward: 5 0 -CACGCTTTCTGTCTACTGA-3 0 and TNF- α Reverse 5 0 -GGACTCCGTGATGTCTAAGT-3 0 ; TSP-1 For-ward: 5 0 -CTTTGCTGGTGCCAAGTGTA-3 0 and TSP-1Reverse: 5 0 -CGACGTCTTTGCACTGGATA-3 0 ; TRB3 For-ward: 5 0 -TGATGCTGTCTGGATGACAA-3 0 and TRB3Reverse: 5 0 -GTGAATGGGGACTTTGGTCT-3 0 ; VCAM-1Forward: 5 0 -GAAGCCGGTCATGGTCAAGT-3 0 andVCAM-1 Reverse: 5 0 -GACGGTCACCCTTGAACAGTTC-3 0 ; VEGF Forward: 5 0 -GAGAATTCGGCCCCAACCATGAACTTTCTGCT-3 0 and VEGF Reverse: 5 0 -GAGCATGCCCTCCTGCCCGGCTCACCGC-3 0 ; beta-actin Forward: 5 0 -TGTGCTATGTTGCCCTAGACTTC-3 0 and beta-actin Reverse: 5 0 -CGGACTCATCGTACTCCTGCT-3 0 . Results were analyzed with SDS 2.1software (Applied Biosystems, Foster City, CA, USA)and relative quantification calculated using the 2 − ΔΔ Ctmethod [26]. Statistical analysis Results were expressed as means± standard errors of the mean (S.E.M.) and % of the Control, as indicated.Comparisons between groups were analyzed by the un-paired Student ’ s t  -test, using GraphPad Prism software,Version 5.0. Differences were considered to be signifi-cant at  P   <0.05. Results The HSu-diet as a rat model of prediabetes with impairedglucose tolerance and insulin resistance Beverage consumption was unchanged between groupsthroughout the experiment. The HSu-treated rats showeda BW profile similar to that of the control animals, duringthe 9 weeks of treatment (Table 1). Similarly, fastingglycemia was unchanged between the two groups (102.90±6.98 vs 96.71±4.49 mg/dl) (Figure 1A); however, in thefed state, blood glucose levels were significantly elevatedin the HSu group compared to Control (162.90±26.54  vs 126.80±13.56 mg/dL; p<0.05) (Figure 1B). During aGTT, the HSu-treated rats showed significantly increasedblood glucose levels when compared with those of theControl group (215.50±19.64  vs  159.00±5.83 mg/dl; p<0.05), 60 min. after the injection of glucose. This differ-ence persisted until the 120 min., when the blood glucoseconcentrations returned to basal levels in the Controlgroup, but remained significantly elevated in the HSu-treated group (168.80±10.64  vs  121.40±3.69 mg/dL; p<0.01) (Figure 1C).Serum fasting insulin concentration in the HSu-treated rats was significantly elevated when comparedwith the Control animals (10.83 ± 1.00  vs  3.74 ± 1.84  μ g/L; p < 0.001) (Figure 1D). A significantly higher value(p < 0.01) of the HOMA-IR index was found in theHSu-treated group, when compared with the Control(Figure 1E). Insulin sensitivity was assessed by the ITT(Figure 1F). The blood glucose levels 120 min. after in-sulin injection, were significantly elevated in the HSu-treated group than in the Control one (63.86 ± 14.78  vs 37.17 ± 7.03 mg/dL; p < 0.01). TRB3 mRNA expression,in the cardiac muscle tissue, was unchanged in bothgroups (data not shown). Table 1 Body, heart and left ventricle weights, bloodpressure, heart rate and lipid profile Parameter Control group(n=7)HSu group(n=7) Body, heart and LV weights BW (g) 390.40± 8.45 397.40± 8.71HW (g) 0.89 ± 0.09 1.10 ± 0.01*LVW (g) 0.45 ± 0.02 0.53 ± 0.02***HW/BW (g/kg) 2.14 ± 0.23 2.62 ± 0.07LVW/BW (g/kg) 1.07 ± 0.02 1.28 ± 0.04** Blood pressure and heart rate SBP (mmHg) 122.00± 1.79 121.60± 1.10DBP (mmHg) 100.10± 1.55 98.40 ± 1.32MBP (mmHg) 108.80± 1.27 109.80± 1.23HR (beats/min) 356.70± 1.00 368.90± 8.14 Lipid profile  TGs (mg/dl) 68.14 ± 9.96 143.10± 23.23* TC (mg/dl) 63.71 ± 0.94 58.25 ± 3.44 Data are expressed as mean±SEM, *P<0.05; ** P<0.01; ***P<0.001 versusControl. BW: body weight; DBP: diastolic blood pressure; HR: heart rate; HW:heart weight; LVW: left ventricular weight; MBP: mean blood pressure; SBP:systolic blood pressure; TC: total cholesterol; TGs: triglycerides. Nunes  et al. Cardiovascular Diabetology   2013,  12 :44 Page 4 of 11http://www.cardiab.com/content/12/1/44
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