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Improved Islet Morphology after Blockade of the Renin-Angiotensin System in the ZDF RatChristos Tikellis,

1Peter J. Wookey,

2Riccardo Candido,

1Sof Andrikopoulos,

3Merlin C. Thomas,

1

and Mark E. Cooper1

The renin-angiotensin system (RAS) has an importantrole in the endocrine pancreas. Although angiotensin IIhas significant effects on cell proliferation and apopto-sis, the contribution of the RAS to changes in isletstructure and function associated with type 2 diabetesis yet to be defined. This study examined the specificeffects of RAS blockade on islet structure and functionin diabetes. Thirty-six male Zucker diabetic fatty (ZDF)rats, 10 weeks of age, were randomized to receive theangiotensin-converting enzyme inhibitor perindopril (8mg/l in drinking water; n � 12), irbesartan (15 mg/kg viagavage; n � 12), or no treatment (n � 12) for 10 weeks.Results were compared with lean littermates (ZL) (n �12) studied concurrently. ZDF rats had increased intra-islet expression of components of the RAS correlatingwith increased intraislet fibrosis, apoptosis, and oxida-tive stress. Disordered islet architecture, seen in ZDFrats, was attenuated after treatment with perindopril orirbesartan. Islet fibrogenesis was also diminished, asmeasured by picrosirius staining and expression of col-lagens I and IV. Gene expression of transforming growthfactor-�1 was increased in the ZDF pancreas (ZL, 1.0 �0.1; ZDF, 2.0 � 0.3; P < 0.05) and reduced after blockadeof the RAS (ZDF � P, 1.3 � 0.2; ZDF � I, 1.5 � 0.1; vs.ZDF, both P < 0.05). Improvements in structural param-eters were also associated with functional improve-ments in first-phase insulin secretion. These findingsprovide a possible mechanism for the reduced incidenceof new-onset diabetes that has been observed in clinicaltrials of RAS blockade. Diabetes 53:989–997, 2004

Activity of the local renin-angiotensin system(RAS) is an important determinant of structureand function in a range of organs, including theheart, kidneys, adrenals, and gonads (1). An

intrinsic RAS has also been demonstrated in the endocrinepancreas (2,3). Human �-cells express both the angioten-sin II (Ang II) type 1 receptor (AT1) and pro-renin genes

(4). Short-term infusion of Ang II impairs first-phaseinsulin release, possibly through changes in intraisletblood flow (5). However, the effects of chronic exposure inthe pancreas to Ang II are largely unknown.

In most tissues, chronic exposure to Ang II increasesoxidative stress (6), activates fibrogenesis (7), and pro-motes apoptosis (8). Each of these processes has alsobeen implicated in the progressive loss of �-cell functionobserved type 2 diabetes (9). In particular, the mainte-nance of the specialized islet architecture and regulationof �-cell number through proliferation/neogenesis andapoptosis are important determinants of islet function(10,11). In type 2 diabetes, �-cell mass is reduced duringthe early stages of diabetes and declines further withdisease progression (12). A number of factors seem toinfluence �-cell loss, including functional overload as aresult of chronic hyperglycemia, free fatty acids, oxidativestress, and amylin (islet amyloid polypeptide) (13,14).Although Ang II is known to have significant effects on cellproliferation (15) and apoptosis (8), the contribution ofthe RAS to the dynamic regulation of islet structure andfunction is yet to be defined.

Recent evidence points to impaired islet function as themajor determinant of oral glucose tolerance (16). At leastinitially, reduced glucose sensitivity in islet cells seems topredominate over insulin resistance in the genesis ofimpaired tolerance to oral glucose (16). Several largeclinical trials (e.g., Heart Outcomes Prevention Evaluation[HOPE] and Losartan for Interventions for Endpoints inHypertension [LIFE]) (17,18) have demonstrated thatblockade of the RAS protects against the development ofdiabetes in “at-risk” patients with hypertension. Thesechanges have largely been attributed to improvements inperipheral insulin sensitivity. However, the degree ofprotection against new-onset diabetes shown in thesestudies suggests a more profound influence on the patho-genesis of type 2 diabetes. We hypothesized that blockadeof the RAS may also attenuate the deleterious actions ofAng II on pancreatic islet structure and function thatcontribute to the progression of �-cell dysfunction andultimately to the development of diabetes. This studyaimed to determine the effect of chronic blockade of theRAS on islet cell structure and function associated withdiabetes, using the well-characterized model of non–insu-lin-dependent diabetes, the genetically obese leptin recep-tor–deficient (fa/fa) Zucker diabetic fatty (ZDF) rat.

RESEARCH DESIGN AND METHODS

Thirty-six male obese ZDF rats, 10 weeks of age, were randomized to theangiotensin-converting enzyme (ACE) inhibitor perindopril (8 mg/l in drinking

From the 1Danielle Alberti Memorial Centre for Diabetic Complications, BakerMedical Research Institute, Melbourne, Victoria, Australia; the 2HowardFlorey Institute of Experimental Physiology and Medicine, University ofMelbourne, Parkville, Melbourne, Victoria, Australia; and the 3Department ofMedicine, Royal Melbourne Hospital, Parkville, Victoria, Australia.

Address correspondence and reprint requests to Professor Mark E. Cooper,Baker Medical Research Institute, P.O. Box 6492, Melbourne, VIC 8008,Australia. E-mail: [email protected].

Received for publication 16 September 2003 and accepted in revised form23 December 2003.

ACE, angiotensin-converting enzyme; Ang II, angiotensin II; AT1, Ang II type1 receptor; DAB, 3,3�-diaminobenzidine; PCNA, proliferating cell nuclearantigen; RAS, renin-angiotensin system; TGF-�, transforming growth factor-�;TUNEL, transferase-mediated dUTP nick-end labeling.© 2004 by the American Diabetes Association.

DIABETES, VOL. 53, APRIL 2004 989

water), the AT1 receptor antagonist irbesartan (15 mg/kg via gavage), or notreatment for 10 weeks. Previous studies have demonstrated that the doses ofperindopril and irbesartan used in this study are associated with end-organprotection in diabetic animals (19,20). ZL rats were studied concurrently andacted as further controls. The end point of 20 weeks was selected as insulinresistance, hyperinsulinemia, hyperglycemia, and islet fibrosis all are ob-served in the ZDF model at this time point (21). All animal procedures werein accordance with guidelines set by the National Health and MedicalResearch Council of Australia.Measurement of physiological and biochemical parameters. At 20 weeksof age, the following parameters were measured in all groups: body weight;systolic blood pressure measured by tail-cuff plethysmography in conscious,prewarmed rats; and GHb measured by high-performance liquid chromatog-raphy (CLC330 GHb Analyser; Primus, Kansas City, MO) (22). After theperformance of intravenous glucose tolerance testing (see below), rats werekilled using a fatal dose of anesthetic. The pancreas was rapidly dissected outand bisected longitudinally, with one half snap frozen in liquid nitrogen andstored at �80°C before use and the other half fixed in 4% paraformaldehydeand embedded in paraffin.Intravenous glucose tolerance test. To assess the first-phase insulinresponse in ZDF rats, we performed glucose tolerance testing (23). Animalswere fasted overnight before the test was commenced. Rats were anesthetizedwith an intraperitoneal injection of sodium pentobarbitone (Nembutal, RhoneMerieux, Australia). The left carotid artery was then cannulated, using aSilastic brand catheter (0.012-inch inside diameter; Dow Corning, Midland,MI) filled with heparinized saline (20 units/ml), and the animals were allowedto stabilize for 20 min. A bolus of glucose at 1 g/kg was injected, using asyringe attached to the cannula, through the carotid artery, and 200 �l ofblood was sampled at 0, 2, 5, and 10 min. The syringe and cannula wereflushed with saline between sampling. Plasma insulin was measured using arat-specific radioimmunoassay kit (Linco Research, El Paso, IL). Plasmaglucose was measured using a glucose analyzer (YSI, Yellow Springs, OH).Picrosirius red staining. Four-micron paraffin sections were prepared from4% paraformaldehyde-fixed, paraffin-embedded rat pancreas. Sections werestained with 0.1% sirius red (Direct red 80; Fluka Chemika, Buchs, Switzer-land) in saturated picric acid (picrosirius red) for 1 h and mounted.Immunohistochemistry. The expression of the �-cell marker proinsulin IIwas examined by immunohistochemistry using an anti–proinsulin II antibody(1:1,000; Biogenesis, Technology RD, Poole, U.K.). Slides were then incubatedwith the primary antibody for 1 h at room temperature. After washing, asecondary antibody (1:500, biotin-conjugated goat anti-rabbit IgG; Dako,Copenhagen, Denmark) was applied for 30 min at room temperature.

For assessing the activity of the RAS in islet sections, staining wasperformed for ACE (1:500; Chemicon, Temecula, CA) and AT1 receptor (1:250;Santa Cruz Biotechnology, Santa Cruz, CA). Recent studies suggest that thenovel enzyme ACE2 is involved in the regulation of the local RAS and may alsobe modified in diabetes (24). Therefore, sections were stained for ACE2 usinga primary antibody donated by Millennium Pharmaceuticals (Cambridge,Boston, MA).

Islet expression of type I and IV collagens was assessed using a polyclonalgoat anti-human type I collagen antibody (diluted 1:100; Southern Biotech,Birmingham, AL) and a polyclonal goat anti-human type IV collagen antibody(diluted 1:200; Southern Biotech). Pretreatment of the sections to be stainedfor collagen phenotypes involved serial incubations with 0.4 and 0.2% pepsinconsecutively (Sigma, St. Louis, MO). After nonspecific blocking in 1% normalhorse serum, sections were incubated with the primary antibodies overnightat 4°C. Biotinylated horse anti-goat immunoglobulin (diluted 1:500; VectorLaboratories, Burlingame, CA) was then applied as a secondary antibody.

Ang II exerts many of its profibrotic effects via the upregulation oftransforming growth factor-� (TGF-�) (25). Islet expression of TGF-� waslocalized using anti–TGF-� antibody (1:500, Santa Cruz Biotechnology). Theprimary antibody was incubated on slides for 1 h at room temperature. Thesecondary antibody (1:500, biotin conjugated goat anti-rabbit IgG; Dako) wasthen applied for 60 min.

Previous studies have shown that islet cell injury in the ZDF rat isassociated with increased NO synthase activity and the accumulation ofnitrate/nitrite within the islet (26). As a marker of nitrative stress, we stainedfor the presence of nitrotyrosine within the islets using a polyclonal rabbitanti-nitrotyrosine antibody (1:100; Upstate Biotechnology, Waltham, MA) for60 min. The secondary antibody (1:125, multilink biotin-conjugated swineanti-goat mouse rabbit immunoglobulin; Dako) was then applied for 30 min.

Amylin (islet amyloid polypeptide) has a number of effects in the rodentislet, including growth promotion and paracrine effects on insulin secretion(27). However, unlike human diabetes, amyloid deposits are not observed inrodent models. The ZDF islet is associated with a progressive reduction instaining for amylin, correlating with �-cell loss and the onset of diabetes. The

presence and distribution of amylin was determined by immunohistochemis-try, performed on pancreas sections using an amylin antibody as previouslydescribed (28). Slides were incubated with the primary antibody for 1 h. Thesecondary antibody (1:500, biotin-conjugated goat anti-rabbit IgG; Dako) wasthen applied for 30 min.

Specific immunohistochemical staining was detected using the standardavidin-biotin complex (Vector Laboratories) method. After thorough washing,the final detection step was carried out using 3,3�-diaminobenzidine (DAB;Sigma) as the chromogen. Sections were lightly counterstained with hema-toxylin and mounted.

All sections were analyzed for staining using light microscopy (OlympusBX-50; Olympus Optical, Tokyo, Japan) and digitized using a high-resolutioncamera (Fujix HC-2000; Fujifilm, Tokyo, Japan). Digitized images were thencaptured and evaluated using an image analysis system (AIS; Imaging Re-search, St. Catherines, Ontario, Canada) coupled to an IBM Pentium IIIcomputer. Semiquantitative assessment of islet proteins was performed bydetermining the percentage proportion of area or number of cells per isletsection occupied by the brown (DAB) staining within each islet (�20objective). A total of 12–20 islets per rat pancreas (n � 8 rats/group) wereanalyzed.

Pancreatic �-cell mass was estimated by multiplying the mean density ofstaining for proinsulin in the islet sections (estimated over 12–20 islets asdetailed above) multiplied by the mean islet area per area of pancreas (fivesections per organ). This was expressed in arbitrary units adjusted for thepancreatic wet weight for individual animals.Islet cell apoptosis and proliferation. Islet cell apoptosis was determinedby transferase-mediated dUTP nick-end labeling (TUNEL) staining. Cell deathwas identified by 3� in situ end labeling of fragmented DNA with biotinylateddeoxyuridine-triphosphate (29). After digestion with Proteinase K (2 �g/ml;Sigma), sections were consecutively washed in TdT buffer (0.5 mol/l cacody-late [pH 6.8], 1 mmol/l cobalt chloride, 0.15 mol/l NaCl) and then incubated at37°C with TdT (25U; Boehringer-Mannheim, Mannheim, Germany) and biotin-ylated dUTP (1 nmol/�l). After washing in TB buffer (300 mmol/l NaCl, 30mmol/l sodium citrate) to terminate the reaction, incorporation of biotinylateddUTP was detected by a modification of the avidin-biotin complex method(30). Horseradish peroxidase–conjugated streptavidin-biotin complex wasapplied (Vector Laboratories). The sections were then developed using DABas the chromogen and lightly counterstained with hematoxylin and mountedin dePex (BDH Laboratory, Poole, England). A mouse spleen with increasedapoptosis was used as a positive control for TUNEL staining.

Intraislet proliferation was assessed by immunohistochemical staining forproliferating cell nuclear antigen (PCNA) using a monoclonal anti-PCNAantibody (1:100; Dako) that had been biotinylated using the Dako Ark system.Specific immunohistochemical staining was detected using the streptavidinhorseradish peroxidase and DAB as the chromogen. All sections wereanalyzed for staining using light microscopy and digital analysis as detailedabove. Semiquantitative assessment of intraislet proliferation was performedby determining the number of PCNA-positive cells per islet section. A total of12–20 islets per rat pancreas (n � 8 rats/group) were analyzed. Results wereexpressed as the mean number of cells staining positively for PCNA per isletsection. To determine the presence of proliferating �-cells, we performed dualstaining for PCNA and insulin by first undertaking immunohistochemistry forPCNA. Before counterstaining and dehydration, the sections were subjectedto immunohistochemistry for insulin as described above, using alkalinephosphatase (blue, alkaline phosphatase substrate kit III; Vector) as thechromogen. Semiquantitative assessment of �-cell proliferation was per-formed by determining the number of cells per islet section staining positivelyfor both proinsulin and PCNA. Again, a total of 12–20 islets per rat pancreas(n � 8 rats/group) were analyzed. Results were expressed as the meannumber of cells staining positively for PCNA and insulin per islet section.Real-time quantitative RT-PCR. Gene expression of ACE, ACE2, TGF-�,AT1 receptor, proinsulin, and amylin in pancreas extracts was determinedusing real-time quantitative RT-PCR performed using the TaqMan system (ABIPrism 7700; Perkin-Elmer, PE Biosystems, Foster City, CA), as previouslydescribed (24). Total RNA was isolated from snap-frozen pancreatic tissueafter homogenization using the Ultra-Turrax (Janke & Kunkel IKA, Labortech-nik, Germany) in Trizol (Life Technologies, Gaithersburg, MD). cDNA wasthen synthesized with a reverse-transcriptase reaction carried out usingstandard techniques (Superscript First Strand Synthesis System for RT-PCR;Life Technologies) with random hexamers, dNTPs, and total RNA. Forassessing genomic DNA contamination, controls without reverse transcrip-tase were included. The oligonucleotides and probes (Table 1) were designedusing the software program Primer Express (PE Applied Biosystems).

The RT-PCR took place with 500 nmol/l forward and reverse primerand 50 nmol/l FAM/TAMRA or FAM/MGB probe and VIC/TAMRA 18S

IMPROVED ISLET MORPHOLOGY IN THE ZDF RAT

990 DIABETES, VOL. 53, APRIL 2004

ribosomal probe, in Taqman universal PCR master mix (PE Biosystems). Eachsample was run and analyzed in triplicate.Statistical analysis. Data are shown as means � SE. Incremental area underthe insulin curve was determined using the trapezoidal rule with subtractionof the basal values. Differences between mean values for variables withinindividual experiments were compared statistically by two-way ANOVA.Comparisons were performed using Statview SE (Brainpower Calabasas). P �0.05 was viewed as statistically significant. For the analysis of RT-PCR results,expression values for ZL animals were given an arbitrary value of 1. Resultsfrom ZDF study groups were expressed as a ratio compared with this controlgroup.

RESULTS

Animal model. ZDF animals were heavier than their ZLlittermates (P � 0.01; Table 2). Neither perindopril norirbesartan significantly influenced body weight in ZDFanimals. Systolic blood pressure was also higher in ZDFrats as compared with ZL rats (P � 0.01). Treatment witheither perindopril or irbesartan reduced the systolic bloodpressure to levels even lower than that seen in untreatedZL rats (P � 0.01).Glycemic control. Fasting plasma glucose levels wereelevated in ZDF animals, consistent with the presence ofdiabetes (Table 2). Similarly, GHb levels were elevated,and the first-phase insulin secretion in response to glu-

cose, calculated as the incremental area under the insulincurve between 0 and 10 min after the intravenous injectionof glucose, was significantly reduced in ZDF animals (Fig.1). Treatment with perindopril or irbesartan did not signif-icantly influence fasting hyperglycemia or glycemic con-trol as assessed by GHb. However, both treatmentssignificantly improved first-phase insulin secretion in ZDFanimals (both vs. ZDF, P � 0.05). First-phase insulinsecretion was negatively correlated with the extent of isletcell apoptosis (R � 0.68, P � 0.02) and nitrotyrosinestaining (R � 0.59, P � 0.04) but was not significantlyassociated with gene expression of insulin, amylin, orcomponents of the RAS (all P 0.05).Islet architecture and fibrosis. Islets in untreated ZDFanimals were significantly enlarged with associated isletcell hypertrophy, disarray of islet architecture, and irreg-ular islet boundaries. Treatment with perindopril andirbesartan largely attenuated these changes (Fig. 2). Fibro-sis of the pancreatic islets was significantly increased inZDF rats as demonstrated on picrosirius staining (Fig. 2C).Increased picrosirius staining was apparent both withinthe islet and at the disrupted islet boundary.

Expression of collagen I and IV protein was also signif-icantly increased in untreated ZDF rats, correlating closelywith picrosirius staining (vs. collagen I, r2 � 0.48; vs.collagen IV, r2 � 0.58, both P � 0.01) and with each other(r2 � 0.53, P � 0.01). Both perindopril and irbesartanreduced expression for collagen I and IV protein, althoughperindopril reduced the picrosirius staining to a greaterextent than irbesartan (Table 3).

The profibrotic growth factor TGF-� was also signifi-cantly increased in pancreatic islets from ZDF (Fig. 2D).TGF-� mRNA levels were increased twofold in untreatedZDF animals (ZL, 1.0 � 0.1; ZDF, 2.0 � 0.3; P � 0.05), andprotein expression was increased by nearly 50% (P � 0.05;Table 3). The increase in gene expression of TGF-� seen inthe ZDF animals was largely confined to within the pan-creatic islets, as demonstrated by in situ hybridization(data not shown). As seen with respect to collagen expres-sion, both treatments significantly reduced TGF-� mRNAand protein expression (ZDF P, 1.3 � 0.2; ZDF I, 1.5 �0.1; vs. ZDF, both P � 0.05).RAS. The intraislet expression of components of the RAS

FIG. 1. First-phase insulin response in study groups expressed as theincremental area under the insulin curve for the first 10 min after anintravenous bolus of glucose (1 g/kg, n � 7 per group). P < 0.05 vs. ZL;†P < 0.05 vs. ZDF. Means � SE shown.

TABLE 1Probes and primers used for real-time RT-PCR in pancreasextracts

ACE3� primer 5�-CACCGGCAAGGTCTGCTT5� primer 5�-CTTGGCATAGTTTCGTGAGGAAprobe FAM5�-CAACAAGACTGCCACCTGCTGGTCC

ACE23� primer 5�-CCCTTCTTACATCAGCCCTACTG5� primer 5�-TGTCCAAAACCTACCCCACATATprobe FAM5�-ATGCCTCCCTGCTCATTTGCTTGGT

AT1R3� primer 5�-CGGCCTTCGGATAACATGA5� primer 5�-CCTGTCACTCCACCTCAAAACAprobe FAM5�-CTCATCGGCCAAAAAGCCTGCGT

Proinsulin II3� primer 5�-TGGTTCTCACTTGGTGGAAGCT5� primer 5�-GACATGGGTGTGTAGAAGAATCCprobe FAM5�-CCCACACACCAGGTAG

Amylin3� primer 5�-GCCAGCTGTTCTCCTCATCCT5� primer 5�-GGTACCACTTCCGACAGGTGTAGprobe FAM5�-TCGGTGGCACTCGGCCACTTG

TGF-�13� primer 5�-TTGCCCTCTACAACCAACACAA5� primer 5�-GGCTTGCGACCCACGTAGTAprobe FAM5�-CCGGGTGCTTCCGCATCACC

TABLE 2Physiological and biochemical parameters of the study groups

n

Bodyweight

(g)SBP

(mmHg)

Fastingglucose(mmol/l) GHb (%)

ZL 10 348 � 7 109 � 4 4.8 � 0.3 4.6 � 0.1ZDF 8 447 � 14* 122 � 2* 9.8 � 0.7* 11.0 � 0.8*ZDF P 12 432 � 7* 94 � 2*† 10.0 � 0.8* 10.0 � 0.6*ZDF I 12 434 � 10* 92 � 3*† 8.6 � 0.6* 11.2 � 0.4*

Data are means � SE. SBP, systolic blood pressure. *P � 0.01 vs. ZL;†P � 0.01 vs. ZDF.

C. TIKELLIS AND ASSOCIATES


is shown in Fig. 3. Protein expression of ACE was in-creased at least twofold in islets from untreated ZDFanimals when compared with ZL rats (Table 4). Expres-sion of ACE mRNA in whole pancreas extracts wasincreased to a similar extent (Table 4). Increased expres-sion of ACE mRNA in ZDF animals was strongly correlatedwith collagen IV expression (r2 � 0.51, P � 0.01) andpicrosirius staining (r2 � 0.28, P � 0.05). Treatment withperindopril significantly reduced the expression of ACEmRNA and protein in ZDF animals. Irbesartan also re-duced the expression of ACE, albeit to a lesser extent.

In the pancreas, ACE2 protein was localized to acini andislets, showing a similar distribution to that of ACE (Fig.3). In addition, the expression patterns of ACE and ACE2

mRNA were strongly correlated (r2 � 0.43, P � 0.01). Inuntreated ZDF rats, ACE2 expression was increased atboth a protein and an mRNA level (Table 4). Increasedexpression of ACE2 mRNA in ZDF animals was stronglycorrelated with TUNEL staining (r2 � 0.41, P � 0.01),collagen IV expression (r2 � 0.36, P � 0.01), and picro-sirius staining (r2 � 0.52, P � 0.01). Unlike ACE, theexpression of ACE2 was also correlated with TGF-�1expression. The expression of ACE2 was reduced by bothperindopril and irbesartan. However, the effect of perin-dopril on gene expression did not reach statistical signif-icance (P � 0.07).

Pancreatic staining for the AT1 receptor was weak in theislets of ZL animals, and expression was localized to ductsand vessels (Fig. 3C). Untreated ZDF animals had greaterislet-specific staining for AT1 receptor (Fig. 3F) comparedwith ZL rats. Pancreatic gene expression of AT1 receptormRNA was also significantly greater in ZDF rats (Table 4).Again both treatments significantly reduced the expres-sion of AT1 receptor protein in islets and AT1 receptormRNA (Table 4).Proinsulin and �-cell mass. Immunohistochemical stain-ing for �-cell marker proinsulin II was strong and intensein ZL islets (Fig. 4A). By comparison, staining for proinsu-lin in ZDF islets was diffuse, with focal trapping of �-cellsas “islands” within matrix boundaries (Fig. 4C) associatedwith an overall reduction in the percentage of proportionalarea staining positively for proinsulin (ZL, 54.0 � 1.8; ZDF,23.0 � 1.9; P � 0.01). This reflected increases in interstitialmaterial (predominantly collagen) in the islet and anoverall increase in islet size. Treatment with perindopril orirbesartan significantly increased staining density for pro-insulin (ZDF P, 42.7 � 1.3; ZDF I, 45.0 � 1.4; vs. ZDF,P � 0.01). This was largely coincident with the reductionin islet fibrosis.

Pancreatic �-cell mass was estimated by multiplying themean islet density of staining for proinsulin by the meanislet area per area of pancreas (five sections per organ).This was expressed in arbitrary units adjusted for thepancreatic wet weight for individual animals. ZDF rats hadmore than twice the �-cell mass of ZL animals (ZL, 71 � 6;ZDF, 194 � 25; P � 0.05). Blockade of the RAS furtherincreased total pancreas insulin content (ZDF P, 322 �35; ZDF I, 347 � 39; vs. ZDF, P � 0.05).

Consistent with hyperinsulinemia observed in thismodel, the expression of proinsulin II mRNA in pancreasextracts was also significantly increased in ZDF rats (ZL,1.0 � 0.2; ZDF, 10.6 � 3.2; P � 0.05). Treatment withperindopril or irbesartan was associated with a reductionin proinsulin II mRNA in pancreas extracts (ZDF P,2.2 � 0.8; ZDF I, 3.9 � 0.6; vs. ZDF, P � 0.05).

FIG. 2. Staining for picrosirius red (left) and TGF-�1 (right) in isletsfrom ZL rats (A and B), untreated ZDF rats (C and D), ZDF rats treatedwith perindopril (E and F), and ZDF rats treated with irbesartan (G

and H). Magnification �850 in A and B, �430 in C–H. Arrow denotesirregular islet boundaries. Color reproduction sponsored by Sanofi-Synthelabo, Paris, France.

TABLE 3Intraislet fibrosis and TGF-�1 quantified by immunohistochemistry

n Picrosirius Collagen I Collagen IV TGF-�1

ZL 8 2.5 � 0.1 14.0 � 0.8 14.2 � 0.8 11.0 � 0.7ZDF 8 13.7 � 0.8* 24.0 � 0.9* 24.4 � 0.9* 16.5 � 1.3*ZDF P 8 3.5 � 0.2*† 16.8 � 0.9† 16.9 � 0.9† 6.3 � 0.6*†ZDF I 8 7.5 � 0.5*† 16.2 � 0.6† 16.2 � 0.6† 4.5 � 0.3*†

Data are proportional area (%) of each islet occupied by cells stainingpositively for specific islet proteins, shown as means � SE. *P � 0.05vs. ZL; †P � 0.05 vs. ZDF.



Amylin. Like the expression of proinsulin II, staining foramylin in ZDF islets was less intense and diffusely distrib-uted throughout the islet (Fig. 4D). Treatment with perin-dopril or irbesartan increased the proportional areastaining for amylin protein, again reflecting the associatedreduction in interstitial matrix. However, pancreatic amy-lin gene expression was significantly increased in ZDF rats(ZL, 1.0 � 0.1; ZDF, 12.8 � 2.3; P � 0.05). Notably, the levelof expression of amylin mRNA was also closely correlatedwith the expression of proinsulin mRNA (R2 � 0.8) in ZDFanimals. Both treatments reduced the pancreatic amylinmRNA, although not to control levels (ZDF P, 7.7 � 1.2;ZDF I, 4.8 � 0.7; vs. ZDF, P � 0.05). The percentagereduction in amylin protein and mRNA achieved afterblockade of the RAS was greater than the reduction instaining for proinsulin II.Apoptosis and proliferation. TUNEL staining was usedto identify apoptotic cells within islet boundaries. Intra-islet cell death was significantly greater in ZDF rats, inwhich there was approximately two to three apoptoticcells per islet section compared with infrequent apoptotic

FIG. 3. Expression of ACE (left), ACE2(middle), and AT1R (right) in isletsfrom ZL rats (A–C), untreated ZDF rats(D–F), ZDF rats treated with perindo-pril (G–I), and ZDF rats treated withirbesartan (J–L). Magnification �850 inA and B, �430 in C–L. Color reproduc-tion sponsored by Sanofi-Synthelabo,Paris, France.

TABLE 4Expression of components of the RAS as quantified by immuno-histochemistry and real-time RT-PCR

n ACE ACE2 AT1R

Immunohisto-chemistry*

ZL 8 10.9 � 1.2 9.2 � 0.8 3.8 � 0.6ZDF 8 28.3 � 2.6* 51.0 � 2.0* 11.0 � 0.5*ZDF P 8 3.2 � 0.3*† 18.0 � 0.1*† 4.8 � 0.4†ZDF I 8 8.6 � 0.9† 15.5 � 1.0*† 3.1 � 0.2*†

Real-timeRT-PCR†

ZL 8 1.0 � 0.2 1.0 � 0.2 1.0 � 0.2ZDF 8 2.2 � 0.2* 2.0 � 0.3* 1.75 � 0.2*ZDF P 8 1.5 � 0.1*† 1.5 � 0.1† 1.1 � 0.1†ZDI I 8 1.6 � 0.2*† 1.1 � 0.1† 1.0 � 0.1†

Data are proportional area (%) of each islet occupied by cells stainingpositively for specific islet proteins, shown as means � SE (forimmunohistochemistry), and data are results from ZDF study groupsexpressed as a ratio compared with ZL shown as mean expression �SE (for real-time RT-PCR). *P � 0.05 vs. ZL; †P � 0.05 vs. ZDF.



cells seen in ZL islets (ZL, 0.3 � 0.1/islet; ZDF, 2.5 �0.3/islet; P � 0.01). Blockade of the RAS reduced TUNELstaining within islet cells to control levels (ZDF P, 0.9 �0.1; ZDF I, 0.6 � 0.1; both vs. ZDF, P � 0.01).

There was no significant difference in the number ofcells staining positively for the PCNA marker between theZL and ZDF islets (Fig. 5). However, blockade of the RASsignificantly increased the number of PCNA-positive stain-ing within islet boundaries compared with both ZL andZDF animals. This increase was almost totally explainedby an increase in cells staining positively for both insulinand PCNA (Fig. 5).Nitrative stress. Within the pancreatic islets of ZDF rats,there were increased levels of oxidative stress as mea-sured by percentage of proportional staining for nitroty-rosine (Fig. 6). Staining for nitrotyrosine was increasedmore than fourfold in ZDF compared with ZL rats (ZDF,

30.1 � 2.7; ZL, 7.3 � 0.7; P � 0.01). Blockade of the RASwith perindopril or irbesartan significantly reduced stain-ing for nitrotyrosine (ZDF P, 10.1 � 0.9; ZDF I, 7.0 �0.7; vs. ZDF, P � 0.01). Nitrotyrosine staining was stronglycorrelated with islet fibrosis (vs. picrosirius, R2 � 0.82,P � 0.01), apoptosis (R2 � 0.62, P � 0.01), and staining forproinsulin (r � �0.72, P � 0.01). Increased expression ofthe AT1 receptor mRNA in the pancreas of ZDF rats wasalso strongly correlated with staining for nitrotyrosine(R2 � 0.45).

DISCUSSION

The development of diabetes in the ZDF rat is associatedwith progressive histopathological changes in pancreaticislets, including selective loss of �-cells and fibrosis. Wedescribe for the first time the attenuation of these changesafter chronic blockade of the RAS. Previous studies haveattributed progressive islet damage seen in this modelto chronic hyperglycemia (glycotoxicity) (21). Althoughtreatment with ACE inhibitors or AT1 receptor antagonistsimproves peripheral insulin sensitivity and glucose dis-posal in ZDF rats (31), the beneficial effects on isletmorphology described here were independent of anydirect effects on glycemic control. These findings suggestthat activation of the local RAS may represent an indepen-dent mechanism for progressive islet damage in the ZDFmodel. In addition, these findings provide a possibleexplanation for the markedly reduced incidence of newonset of diabetes observed in clinical trials involving ACEinhibitors and Ang II receptor antagonists (17,18).

The mechanisms that lead to activation of the RAS in thepancreas of ZDF rats have not been established. It haspreviously been demonstrated that chronic hyperglycemiaand hyperlipidemia activate the local RAS (32). Evenbefore the development of diabetes, ZDF rats have in-creased pressor responsiveness to Ang II (33). In thekidney, this may be related to increased AT1 receptorabundance and function (34). Consistent with this finding,expression of AT1 receptor protein was increased morethan threefold in the islets of ZDF rats, correlating withfibrosis and �-cell loss. Studies in obese patients suggestthat weight gain itself activates the RAS (35). Pancreaticinflammation may also result in upregulation of the localRAS as demonstrated in animal models of pancreatitis

FIG. 4. Dense immunostaining for proinsulin (left) and amylin (right)in ZL islets (A and B) and diffuse staining in untreated ZDF islets (C

and D). Increased staining density after blockade of the RAS withperindopril (E and F) and irbesartan (G and H). Magnification �850 inA and B, �430 in C–H.

FIG. 5. Increased number of islet cells per section staining positivelyfor PCNA in ZDF rats treated with perindopril and ZDF rats treatedwith irbesartan is explained by an increase in PCNA-positive �-cells.*P < 0.01 vs. ZL; †P < 0.05 vs. ZDF. Means � SE shown.



(36). However, we saw no evidence of insulitis in thismodel. In addition, in the ZDF model, early extracellularmatrix remodeling in the kidney seems to be independentof any inflammatory process (37). Finally, hypertension inthe ZDF rat may also contribute to activation of the RASand potentially to progressive �-cell dysfunction in thismodel (38). It is conceivable that excellent blood pressurecontrol after blockade of the RAS may have contributed totheir observed benefit. If this were true, then other anti-hypertensive agents, such as calcium channel blockers,may also confer benefits on islet function and structure.However, it should be noted that previous studies with thecalcium channel blocker verapamil showed a paradoxicalworsening of hyperinsulinemia despite an equivalent anti-hypertensive effect to captopril (38). Phenomenologically,this is similar to the differential effects of blockade of theRAS on proteinuria in this model (39).

The maintenance of the specialized architecture of thepancreatic islet is important for continuing function. Dis-ruption of contacts between �-cells reduces the secretoryefficiency of islets (40). Loss of cell-to-cell communicationassociated with increased intraislet fibrosis may also pro-mote islet cell apoptosis (41). The RAS has been linked toincreased fibrosis in a variety of tissues, including theheart (42), kidney (43), and liver (44). Locally generatedAng II is thought to activate the AT1 receptor, resulting inupregulation of the fibrogenic cytokines and growth fac-tors, including TGF-�1 (25). In addition to stimulating thedeposition of increased amounts of extracellular matrix,TGF-�1 is known to regulate cell growth, differentiation,and function in the pancreas (45). We report here theincreased expression of TGF-� mRNA and protein in the

islets of ZDF rats (Table 3). TGF-� has been previouslyassociated with fibrosis after pancreatitis (36,46) andautoimmune diabetes (47). However, this is the first de-scription of increased islet expression of TGF-� associatedwith a model of type 2 diabetes. In other tissues, blockadeof the RAS seems to result in reduced fibrosis throughinhibition of TGF-�1 (20). In models of rodent pancreatitis,blockade of the RAS results in lower levels of TGF-� andfibrosis (36). It is conceivable that the reduction in isletfibrosis observed in this study after treatment with perin-dopril and irbesartan may be mediated through a similarpathway.

The regulation of islet cell apoptosis and proliferation isalso important in maintaining �-cell mass, particularly inthe setting of concomitant insulin resistance. In particular,the pivotal role of apoptosis has been demonstrated by thedevelopment of diabetes in the animals with partial pan-creatic duodenal homeobox-1 deficiency (an animal modelof maturity-onset diabetes of the young, type 4) (10). Inthis model, increase �-cell apoptosis leads to �-cell deple-tion and an abnormal islet architecture similar to thatdescribed here (10). Although Ang II is known to influencegrowth and proliferation in the endocrine pancreas (5), thecontribution of these changes to islet dysfunction indiabetes has not been previously investigated. In culturedrat renal proximal epithelial cells, Ang II triggers cell deathafter the upregulation of TGF-� (8). In addition, the tubu-lar expression of TGF-� and tubular apoptosis are bothsignificantly increased in the diabetic transgenic(mRen-2)27 rat associated with overactivity of the localRAS (48). Both of these changes can be attenuated afterblockade of the RAS (49). Consistent with these renal

FIG. 6. Immunostaining for nitrotyrosine inZL islets (A), untreated ZDF islets (B),ZDF islets treated with perindopril (C),and ZDF islets treated with irbesartan (D).Magnification �850 in A, �430 in B–D.



findings, we demonstrate increased apoptosis and expres-sion of TGF-� in the ZDF islets, both of which wereprevented after treatment with perindopril or irbesartan.

Previous studies in the ZDF rat have demonstrated thatislet cell proliferation is increased early but is not signifi-cantly different from lean littermates by 12 weeks of age(50). Consistent with this finding, we found no significantdifference between ZL and ZDF rats at 20 weeks of age, atime when diabetes is established. Although the local RAShas independent antiproliferative effects in many tissues(1), an effect on islet cell proliferation and neogenesis hasnot been previously documented. In our experiments, bothperindopril and irbesartan were associated with a signifi-cant increase in intraislet proliferation as demonstrated bypositive staining for PCNA. This increase was almostentirely explained by the presence of proliferating �-cells.Along with reduced apoptosis, these changes may signifi-cantly contribute to enhanced �-cell mass in this modelafter blockade of the RAS. Although a role for the RAS inislet cell regeneration and neogenesis is plausible, furtherresearch is required to delineate its significance in theonset and progression of type 2 diabetes

Pancreatic islets are highly susceptible to oxidativeinjury, owing to low endogenous antioxidant activity (51).The ZDF model is associated with elevated levels ofoxidative stress in the pancreas (52). In addition, pro-oxidant challenge in vivo provokes the onset of diabetes inthe ZDF rat (53). In �-cells, glucose stimulates the produc-tion of reactive oxygen species in islets through proteinkinase C–dependent activation of NAD(P)H oxidase (54).Hyperglycemia (and, subsequently, advanced glycation andhyperinsulinemia) further increases the generation of reac-tive oxygen species. Ang II also increases tissue NAD(P)Hoxidase activity (55). ACE inhibitors and angiotensin re-ceptor antagonists indirectly inhibit NAD(P)H oxidase bypreventing activation of the AT1 receptor (6). Directeffects on NAD(P)H oxidase activity have also been attrib-uted to some ACE inhibitors and angiotensin receptorantagonists (56). It is also possible that reduction ofoxidative stress (as measured by staining for nitroty-rosine) contributes to the preservation of islet architectureand reduction in fibrosis seen after blockade of the RAS inthe ZDF model. Notably, changes in intraislet oxidativestress in this study were the strongest predictor of isletmorphology and function.

Although islet architecture was maintained after block-ade of the RAS, these agents did not influence long-termglycemic control or protect against the onset of diabetes inthis model. This may be a result of the comparatively lateintervention with RAS blockade used in this study. Al-though this time point may potentially be better repre-sentative of the high-risk patients shown to benefit fromRAS blockade, ZDF rats at 10 weeks of age already havesubstantial �-cell loss and glucose intolerance. It is con-ceivable that an earlier intervention (e.g., from 6 to 7weeks of age) may have proved more beneficial. Indeed,studies using rosiglitazone have suggested that a therapeu-tic window may exist for islet structural and functionalimprovement, its end corresponding with the developmentof persistent hyperglycemia (50). Nonetheless, the first-phase insulin response was significantly improved afterblockade of the RAS, suggesting an improvement in islet

function. Some of this improvement may reflect increased�-cell mass as well as changes in intraislet blood flow (5),improved glucose delivery, and insulin washout. Intraisletamylin may also act as a local inhibitor of stimulated �-cellsecretion (27) and was significantly reduced after block-ade of the RAS in ZDF rats. In addition, the disruption ofthe integrity of contiguous anatomical structures associ-ated with islet hypertrophy in the ZDF rat interferes withcoordinated insulin secretion (57). However, the impor-tance of these functional and structural changes to theimpairment of first-phase insulin release remains to bedetermined.

In summary, the local RAS is upregulated in the islets ofZDF rats associated with the disruption of islet architec-ture, fibrosis, and apoptosis. This is consistent with obser-vations in other tissues, where locally released Ang II hasan important role in the regulation of both organ structureand function. In the ZDF model, blockade of the RASsignificantly attenuated islet damage and augmented �-cellmass, possibly by a reduction in oxidative stress, apopto-sis, and attenuation of profibrotic pathways. These find-ings provide a novel mechanism that could partly explainthe reduced incidence of new-onset diabetes that has beenobserved in clinical trials involving ACE inhibitors andAng II receptor antagonists.

ACKNOWLEDGMENTS

The Division of Diabetic Complications at the BakerMedical Research Institute is supported by grants from theJuvenile Diabetes Research Foundation. M.C.T. is sup-ported by an Australian National Health and MedicalResearch Council biomedical scholarship. R.C. was sup-ported by a grant from the University of Trieste, Italy. S.A.is supported by a career development award from theNational Health and Medical Research Council of Australia.

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