Renin Inhibition Attenuates Insulin Resistance, Oxidative Stress, and ...

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Endocrinology 149(11):5643–5653 Copyright © 2008 by The Endocrine Society doi: 10.1210/en.2008-0070

Renin Inhibition Attenuates Insulin Resistance, Oxidative Stress, and Pancreatic Remodeling in the Transgenic Ren2 Rat Javad Habibi, Adam Whaley-Connell, Melvin R. Hayden, Vincent G. DeMarco, Rebecca Schneider, Susan D. Sowers, Poorna Karuparthi, Carlos M. Ferrario, and James R. Sowers Departments of Internal Medicine (J.H., A.W.-C., M.R.H., V.G.D., R.S., S.D.S., P.K., J.R.S.), Physiology and Pharmacology (J.R.S.), and Nephrology (A.W.-C.) and Diabetes Cardiovascular Center (J.H., A.W.-C., M.R.H., V.G.D., R.S., J.R.S.), University of Missouri-Columbia School of Medicine, Columbia, Missouri 65212; Wake Forest University School of Medicine (C.M.F.), Wake Forest University, Winston-Salem, North Carolina 27157; and Harry S. Truman Veterans Affairs Medical Center (A.W.-C., J.R.S.), Columbia, Missouri 65201 Emerging evidence indicates that pancreatic tissue expresses all components of the renin-angiotensin system. However, the functional role is not well understood. This investigation examined renin inhibition on pancreas structure/function in the transgenic Ren2 rat harboring the mouse renin gene, a model of tissue renin overexpression. Renin is the rate-limiting step in the generation of angiotensin II (Ang II), which stimulates the generation of reactive oxygen species in a variety of tissues. Overexpression of renin in Ren2 rats results in hypertension, insulin resistance, and cardiovascular and renal damage. Young (6 –7 wk old) insulin-resistant male Ren2 and age-matched insulin sensitive Sprague Dawley rats were treated with the renin inhibitor, aliskiren (50 mg/kg䡠d by ip injection), or placebo for 21 d. At 21 d, the Ren2 demonstrated insulin resistance with increased islet insulin, Ang II, and reduced total insulin receptor substrate (IRS)-1, IRS-2, and Akt immunostaining. There was increased islet nicotinamide

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HE AUTOCRINE/PARACRINE ACTIVITY of tissue renin-angiotensin system (RAS) is an important modulator of structure and function of a number of organs including the heart, kidneys, vasculature, and skeletal muscle (1). A recently identified local islet RAS may play an important role in pancreatic physiology and pathophysiology (2– 6). Pancreatic islet ␤-cells express prorenin, renin, angiotensin II (Ang II), and the Ang II type 1 receptor (AT1R) (5, 6). Activation of local RAS is associated with increased oxidative stress in cardiovascular and skeletal muscle tissue in insulinresistant rodents (7, 8). However, it is unknown whether tissue RAS activation causes increased oxidative stress in the First Published Online July 24, 2008 Abbreviations: Ang II, Angiotensin II; AT1R, Ang II type 1 receptor; AUC, area under the curve; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ␤-HAD, ␤-Hydroxyacyl-CoA dehydrogenase; IEI, islet exocrine interface; IPGTT, ip glucose tolerance test; IRS, insulin receptor substrate; NADPH, nicotinamide adenine dinucleotide phosphate; RAS, renin-angiotensin system; Ren2-A, Ren2 treated with aliskiren; Ren2-C, Ren2 control; ROS, reactive oxygen species; SBP, systolic blood pressure; SD, Sprague-Dawley; SD-A, SD treated with aliskiren; SD-C, SD control; TEM, transmission electron microscopy; VVG, Veorhoff-von Gieson. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

adenine dinucleotide phosphate (NADPH) oxidase activity and subunits (p47phox and Rac1) as well as increased nitrotyrosine immunostaining (each P < 0.05). These functional abnormalities were associated with a disordered islet architecture; increased islet-exocrine interface, pericapillary fibrosis, and structurally abnormal mitochondria and content in endocrine and exocrine pancreas. In vivo treatment with aliskiren normalized systemic insulin resistance and islet insulin, Ang II, NADPH oxidase activity/subunits, and nitrotyrosine and improved total IRS-1 and Akt phosphorylation (each P < 0.05) as well as islet/exocrine structural abnormalities. Collectively, these data suggest that pancreatic functional/structural changes are driven, in part, by tissue reninangiotensin system-mediated increases in NADPH oxidase and reactive oxygen species generation, abnormalities attenuated with direct renin inhibition. (Endocrinology 149: 5643–5653, 2008)

pancreas and whether there are functional and structural consequences of increased generation of reactive oxygen species (ROS). Potential sources of ROS include mitochondria, the xanthine oxidase enzyme, and the membrane reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme (7, 8). In this regard, blockade of the RAS using angiotensin receptor (AT1R) antagonists has been shown to attenuate NADPH oxidase activity and reduce ROS and associated pancreatic fibrosis in the Zucker obese insulin-resistant diabetic rat (6) and the skeletal muscle of transgenic Ren2 rats (7). These preliminary findings suggest that tissue NADPH oxidase enzyme activity may play a key role in glucose homeostasis. Activation of the AT1R is known to increase activity of the NADPH oxidase enzyme by translocation of cytosolic subunits (p47phox, p40phox, p67phox, and Rac1) to the plasma membrane and contribute to formation of ROS in a number of tissues (8 –16), including the islet and exocrine pancreas (15–17). The islet and exocrine pancreas have a limited capacity to handle oxidative stress (18), making them quite vulnerable to RAS-mediated oxidative stress (17–20). In this regard increased tissue RAS activity significantly impairs islet blood flow in isolated perfused rat pancreas and inhibits insulin release from mouse islets in response to high glucose, abnormalities improved with RAS blockade (20 –23). Prore-

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Endocrinology, November 2008, 149(11):5643–5653

nin, found in the pancreas (5, 6), is converted into renin, the rate-limiting enzyme in the generation of Ang II. Thus, we hypothesized that inhibition of this enzyme should abrogate any direct effects of renin as well as reduce tissue Ang II levels. The new nonpeptide renin inhibitor, aliskiren, is a potent human and mouse renin inhibitor (24, 25). Because aliskiren shows species specificity for its substrate (only human and mouse renin), aliskiren cannot be studied efficiently in conventional rat models. To circumvent this issue, we used the transgenic (mRen2)27 rat (Ren2), which overexpresses the mouse renin transgene with resultant elevated tissue levels of Ang II (9, 26). This allows for the investigation of inhibition of the rate limiting enzyme in a model of chronic RAS activation, which presumably leads to elevated pancreas Ang II levels. We previously used this model to evaluate the role of elevated cardiac, renal, and skeletal muscle RAS on NADPH oxidase generation of ROS and resulting cardiovascular and renal structural/functional changes (9 –15). We hypothesized that blockade of the RAS with chronic aliskiren treatment would attenuate the deleterious effects of Ang II on pancreatic islet structure and function that contribute to the development of diabetes in the Ren2 rat (9). We further investigated the notion that the beneficial effects of renin inhibition would be mediated, in part, through reductions in membrane NADPH oxidase. To address this hypothesis, we conducted in vivo/ex vivo studies of the effects of 3 wk therapy with aliskiren on islet structure and function in 7- to 9-wk-old Ren2 and littermate Sprague Dawley (SD) controls. This age span represents a time when Ren2 rats are insulin resistant but euglycemic, (9), with increased tissue oxidative stress (9 –15). Materials and Methods Animals and treatments All animal procedures were approved by the University of Missouri animal care and use committees and housed in accordance with National Institutes of Health guidelines. Heterozygous male Ren2 (4 – 6 wk of age) and age-matched male SD rats were randomly assigned to placebo [Ren2 control (Ren2-C) and SD control (SD-C), n ⫽ 5 each] or aliskiren [Ren2 aliskiren (Ren2-A) and SD aliskiren (SD-A), n ⫽ 5 each] 50 mg/kg䡠d ip injection treatment for 21 d.

Systolic blood pressure (SBP) Restraint conditioning was performed before initial blood pressure measurements. SBP was measured in triplicate on separate occasions throughout the day, using the tail-cuff method (student oscillometric recorder, model MC4000 RSP; Harvard Systems, Hatteras Instruments Inc., Cary, NC) before initiation of treatment and on d 19 or 20 before the animals were killed (9 –14).

Intraperitoneal glucose tolerance test (IPGTT) and weight Animals were food restricted overnight before the experiment. On d 21, rats were weighed and gently restrained. A 200-␮l blood sample via tail vein was drawn for insulin (ELISA) and glucose measurement immediately with a glucometer (Dex; Bayer, Elkhart, IN). A dose of dextrose (50% solution, 1 g/kg body weight) was injected ip, and blood was drawn at 15, 30, 45, and 60 min for insulin and glucose determination. Serum was separated and frozen at ⫺80 C until analyzed for insulin (RIA kit; Linco, St. Charles, MO). Insulin resistance index was calculated as the product of areas under the glucose and insulin curves (AUCglucose ⫻ AUCinsulin) as previously described (9).

Habibi et al. • Aliskiren Attenuates Pancreatic Oxidative Stress

Immunohistochemistry quantification of insulin and Ang II Four-micrometer sections of pancreas of SD-C, SD-A, Ren2-C, and Ren2-A were dewaxed in CitriSolv (Fisher Scientific, Pittsburgh, PA) and rehydrated in ethanol series and HEPES wash buffer. The epitopes were retrieved in citrate buffer at 95 C for 25 min. Nonspecific binding sites were blocked by goat blocker for 4 h. Then the sections were incubated with rabbit polyclonal Ang II (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA) and guinea pig polyclonal to insulin (1:70) overnight (Abcam, Cambridge, MA). Then the sections were washed (3 ⫻ 15 min) with HEPES wash buffer and incubated with secondary antibodies, donkey antirabbit, and Alexa flour 647 (Invitrogen, Eugene, OR) for Ang II and goat antiguinea pig Alexa flour 546 for insulin. After washing thoroughly, the sections were incubated with 1:2000 4⬘,6⬘-diamidino-2-phenylindole for 15 min. Eventually the sections were washed and mounted with mowiol. The images were captured and the signals were analyzed as previously described (9, 11). Briefly, slides were checked under a biphoton confocal microscope (Bio-Rad, Hercules, CA), and the Z optimal images of three islets and exocrine tissue were captured with laser confocal microscope imaging system. On each image, equal areas (200 ⫻ 200 pixels) were randomly selected and then quantified and analyzed with MetaVue (Boyce Scientific Inc. Gary Summit, MO). The mean of each animal was calculated, and then the mean of five animals were used in the analysis.

Quantification of insulin receptor substrate (IRS)-1, IRS-2, and Akt in endocrine islet and exocrine tissue Four-micrometer pancreatic sections of SD-C, SD-A, Ren2-C, and Ren2-A were prepared for staining as mentioned above. Then the sections were incubated with rabbit anti IRS-1 1:50 and IRS-2 1:50 overnight (Santa Cruz Biotechnology) and Alex flour donkey antirabbit as secondary antibody. Sections were also incubated with rabbit polyclonal Akt (Ser473) (1:50) and Akt (Thr308) (1:50) (Cell Signaling Technology, Charlottesville, VA), as previously described (9, 11) overnight and 4 h with the secondary antibody, Alexa flour donkey antirabbit 647. The images were captured and signals analyzed as described above.

Western blot analysis Standard Western blot analysis was used to quantify IRS-1 and pAkt as described previously (12). Briefly, the pancreas tissue was homogenized using a glass-on-glass Dounce homogenizer in sucrose homogenization buffer and centrifuged at 1000 ⫻ g to remove connective tissue. Protein concentrations were measured by o-phthaldialdehyde as above. Forty micrograms of total proteins from different samples were loaded in each lane and analyzed via SDS-PAGE under reducing conditions on a 7.5% gel for pAkt and 5% gel for IRS-1. The protein was transferred to a nitrocellulose membrane and blocked in 5% milk in Tris-buffered saline Tween 20 for 30 min. Then the blocks were incubated with antiIRS-1 (C-20), 1:1000 (Santa Cruz), and anti-pAkt (Ser473), 1:1000 (Cell Signaling) in blocker overnight. The blocks were then incubated with antirabbit horseradish peroxidase conjugated, 1:40,000, were developed on the q-dot and the intensity of the bands were quantified with Quantity One software (Bio-Rad).

Mouse renin transcript expression in the pancreas: reverse transcription Total RNA was extracted from snap-frozen pancreas samples from 9-wk-old untreated and aliskiren-treated male Ren2 and SD rats using PureLink Micro-to-Medi RNA purification kit (Invitrogen) (n ⫽ 4 –5/ group). The purity and concentration of RNA extracts were determined spectrophotometrically using a Nanodrop (Thermo Fisher Scientific, Wilmington, DE). Moloney murine leukemia virus reverse transcriptase and random olgio-dT (Promega, Madison, WI) were used to reverse transcribe 200 ng of mRNA to cDNA. The reaction incubated for 60 min at 42 C in an iCycler thermocycler (Bio-Rad). The cDNA samples were then incubated at 95 C for 5 min in the thermocycler to inactivate the reverse transcriptase. A 1.8-␮g aliquot of cDNA was analyzed using PCR.

PCR Reverse transcriptase-generated cDNA encoding mouse ren2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified



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using PCR. GAPDH, a housekeeping gene, was used as an internal standard. The oligonucleotide primer sequences for mouse ren2 were designed in accordance with published mouse and rat DNA sequences for ren2 (accession no. NM_031193) and GAPDH (accession no. NM_017008), respectively (mouse ren2 forward: 5⬘-CAA AGA GGT CTT CCT TGA CTG-3⬘; mouse Ren2 reverse: 5⬘-AAA TCG CCT TGG TAA TGC TCC-3⬘; rat GAPDH forward: 5⬘-ACC ACA FTC CAT GCC ATC AC-3⬘; rat GAPDH reverse: 5⬘-TCC ACC ACC CTG TTG CTG TA-3⬘). Using these primer sets, the expected band sizes for mouse ren2 and GAPDH are 583 and 425 bp, respectively. The experimental conditions for mouse ren2 and rat GAPDH PCRs were: initial denaturation at 94 C for 1 min, 32 cycles of amplification at 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec, and final extension at 72 C for 10 min. A negative control for each set of PCRs contained water instead of the DNA template. All PCR products (10 ␮l) were electrophoretically separated on a 1% agarose gel and then stained with ethidium bromide. A FluoroChem 8800 gel imaging and documentation system (Alpha Innotech, San Leandro, CA) was used to visualize the PCR products.

dewaxed in CitriSolv and rehydrated in ethanol series and HEPES wash buffer. The epitopes were retrieved in citrate buffer at 95 C for 25 min. Nonspecific binding sites were blocked by goat blocker for 4 h. Then the sections were incubated with mouse antihuman complex IV subunit 1 monoclonal antibody, 3 ␮g/ml in 10-fold diluted blocker (Mitosciences, Eugene, OR) or Mitotracker deep-red, 200 nm in 10-fold diluted blocker, (Invitrogen) overnight. After washing with HEPES wash buffer, the sections that were incubated with monoclonal antibody were incubated with 1:300 goat-antimouse Alexa flour 647 (Invitrogen). After 4 h, the slides were washed and incubated with 1:2000 4⬘,6⬘-diamidino-2-phenylindole. After 10 min, the slides were washed and mounted with mowiol. The slides were checked with a laser confocal microscope and a multiphoton confocal system (Carl Zeiss, Thornwood, NY), and images were captured by laser-sharp, enhanced with Adobe Photoshop (San Jose, CA), and the mitochondria were quantified by MetaMorph (Molecular Devices, Downingtown, PA).

NADPH oxidase activity and subunits

Citrate synthase activity, measured as a surrogate for entry into the mitochondrial KREBs cycle, was determined as previously described (12). Briefly, pancreas tissue homogenates were incubated in the presence of oxaloacetate, acetyl-CoA, and dithionitrobenzoic acid. Spectrophotometric detection of reduced dithionitrobenzoic acid at a wavelength of 412 nm served as an index of enzyme activity.

NADPH oxidase activity was determined in plasma membrane fractions of pancreas tissue as previously described (10 –12). Pancreas tissue was harvested, fixed and embedded in paraplast, sectioned, and evaluated for NADPH oxidase subunits p47phox and Rac1 as previously described (9 –14).

Citrate synthase activity

␤-Hydroxyacyl-CoA dehydrogenase (␤-HAD) activity

3-Nitrotyrosine immunostaining To assess pancreas nitrotyrosine content, 4 ␮m of pancreas tissue sections were deparaffinized and rehydrated, and epitopes were retrieved in citrate buffer as previously described (12). Images were analyzed and the signal intensities measured with MetaVue (Boyce Scientific).

Quantification of arterial remodeling Four microns of fixed paraffin sections of SD-C, Ren2-C, SD-A, and Ren2-A were stained with Veorhoff-von Gieson (VVG) as previously described (9 –11). Briefly, images from all cross sections of arteries in approximately 1 cm2 (minimum 20 arteries per animals) were captured and fibrosis quantified by MetaVue (Boyce Scientific).

Transmission electron microscopy (TEM) Tail/body sections of the pancreatic tissue were thinly sliced and placed in primary electron microscopy fixative [2% glutaraldehyde, 2% paraformaldehyde in 0.1 m Na cadylate buffer (pH 7.35)] as previously described (9, 12, 13). Specimens were then stained with 5% uranyl acetate and Sato’s triple lead stain. A TEM (1200-EX; Jeol, Ltd., Tokyo, Japan) was used to view all pancreatic samples.

Mitochondrial quantification The harvested pancreas tissue of SD-C, SD-A, Ren2-C, and Ren2-A were fixed in 3% fresh paraformaldehyde, infiltrated, and embedded in paraplast. The pancreatic tissue of four animals from each treatment group was used for analysis. Four-micrometer sections of pancreas were

␤-HAD activity, as an indicator of mitochondrial fatty acid oxidation, was measured at 37 C in assay buffer containing 0.1 m triethanolamineHCl, 5 mm EDTA, and 0.45 mm nicotinamide adenine dinucleotide hydroxide (pH 7.0) (27). After an initial 2-min absorbance reading at 340 nm, the reaction was initiated by adding 0.1 mm acetoacetyl-CoA, and the rate of disappearance of nicotinamide adenine dinucleotide hydroxide, and the appearance of nicotinamide adenine dinucleotide was measured by change in absorbance every 10 sec for 5 min. Enzyme activity was expressed as nanomoles per gram protein per minute.

Statistical analysis All values are expressed as mean ⫾ se. Statistical analyses were performed in SPSS 13.0 (SPSS Inc., Chicago, IL) using ANOVA with Fisher’s least significant differences as appropriate and student’s t test for paired analysis. Significance was accepted as P ⬍ 0.05.

Results SBP, IPGTT, and weight

At the start and end of the treatment period, there were increases in SBP of the Ren2 when compared with agematched SD controls (Table 1), and by the end of the treatment period, the SBP in Ren2-A were significantly lower than pressures in age-matched, placebo-treated Ren2-C (Table 1). Ren2 rats had higher fasting and 15-, 30-, 45-, and 60-min insulin values and (each P ⬍ 0.05 except for 15 min value was

TABLE 1. Experimental parameters SD-C

Systolic blood pressure (mm Hg) Initial Final ␦ Weight (g) Initial Final ␦ a b

SD-A

Ren2-C

Ren2-A

124.8 ⫾ 3.9 138.8 ⫾ 4.2 14.0 ⫾ 3.8

132.3 ⫾ 2.4 136.9 ⫾ 1.8 4.6 ⫾ 2.0

152.8 ⫾ 7.2a 196.2 ⫾ 3.5a 43.4 ⫾ 3.7a,b

157.4 ⫾ 1.9a 153.6 ⫾ 4.3a,b ⫺3.8 ⫾ 5.1a,b

83.8 ⫾ 6.2 252.6 ⫾ 10.1 168.8 ⫾ 7.0

145.6 ⫾ 4.7a,b 231.8 ⫾ 5.2 86.2 ⫾ 2.8b

107.5 ⫾ 2.2a 249.4 ⫾ 8.7 146.8 ⫾ 12.0

139.8 ⫾ 7.8a,b 232.0 ⫾ 10.8 92.2 ⫾ 8.1b

P ⬍ 0.05 when compared with age-matched SD-C. P ⬍ 0.05 when aliskiren-treated animals are compared with age-matched controls.


µU/ml

A

Insulin 5 4 3 2 1 0

*

* **

*

** 15

30

250

D SD-C

I

I

**

45

60

E

150

Ren2-A

Ren2-C

*

200

SD-A E

*

**

Glucose

B

mg/dl

SD-C SD-A Ren2-C Ren2-A

#

0

I

I

**

100

0

15

30

45

60

E

E

E

C

arbitrary units


Insulin Resistance Index

*

5000 4000 3000 2000 1000 0

**

SD-C

SD-A Ren2-C Ren2-A

Islet Insulin

Average Grey Scale Intensities

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1.2

**

0.8 0.4 0

SD-C

SD-A Ren2-C Ren2-A

FIG. 1. Renin inhibition improves insulin resistance in the Ren2. A and B, Blood glucose and insulin response to ip glucose tolerance test in untreated Ren2 and aliskiren-treated rats compared with SD-C. C, Insulin resistance index is AUCglucose ⫻ AUCinsulin. D, Representative images of immunohistochemistry analysis of pancreas islet (I, Islet; E, exocrine tissue) insulin. E, Average gray-scale intensity measures of D. *, P ⬍ 0.05 when compared with SD-C; #, P ⫽ 0.05 when compared with SD-C; **, P ⬍ 0.05 when Ren2-A is compared with Ren2-C.

P ⫽ 0.05) and 30-min blood glucose (P ⬍ 0.05) compared with SD controls during IPGTT (Fig. 1, A and B). Treatment with aliskiren decreased insulin at 30, 45, and 60 min and glucose at 30 min (each P ⬍ 0.05). Similarly, there were increases in pancreas islet immunostaining of insulin in the placebotreated Ren2, which became similar to that of SD islets with aliskiren treatment (Fig. 1D). Collectively, there was an increase in the insulin resistance index in the Ren2 compared with SD control (P ⬍ 0.05), and this normalized with aliskiren treatment (P ⬍ 0.05) (Fig. 1C). Additionally, there were no significant changes in final total body weights in the Ren2 compared with SD control or treated rats (Table 1). Immunostaining for islet IRS-1, IRS-2, and Akt

There were decreases in islet IRS-1 and -2 levels as determined by immunostaining in Ren2 islets (48.6 ⫾ 4.6 and 24.7 ⫾ 2.5 average gray-scale intensities, respectively) compared with SD controls (61.8 ⫾ 4.7 and 41.1 ⫾ 3.8 average gray-scale intensities, respectively; each P ⬍ 0.05) (Fig. 2, A and D). The levels of islet IRS-1 immunostaining increased to levels comparable with SD islets in the aliskiren-treated Ren2 islets (87.9 ⫾ 10.3 average gray-scale intensities, P ⬍ 0.05) by immunostaining (Fig. 2B) and Western analysis (1.97 ⫾ 0.27 arbitrary units; P ⬍ 0.05) (Fig. 2C). However, aliskiren did not

significantly increase the levels of islet IRS-2 (23.5 ⫾ 1.9 average gray-scale intensities) (Fig. 2D). Similarly, there was a trend to decreased islet Akt levels in the Ren2 (69.3 ⫾ 7.1 average gray-scale intensities) compared with SD controls (94.0 ⫾ 11.4 average gray-scale intensities), and the levels of islet Akt increased with aliskiren treatment (119.4 ⫾ 15.7 average gray-scale intensities, P ⬍ 0.05) (Fig. 3A). There were no significant reductions in islet Ser473 and Thr308 Akt phosphorylation in the Ren2 vs. the SD islets (18.3 ⫾ 1.0 and 80.5 ⫾ 14.3 average gray-scale intensities, respectively) (Fig. 3D). However, there were significant Akt phosphorylation increases at both sites with aliskiren treatment in immunostaining of Ren2 islets (22.1 ⫾ 1.2 and 116.9 ⫾ 8.0 average gray-scale intensities, respectively; P ⬍ 0.05) as well as a trend by Western analysis (3.87 ⫾ 1.42 arbitrary units, P ⬍ 0.1) (Fig. 3, C and D). Renin transgene expression and Ang II immunostaining of islet and exocrine pancreas

Transcript of the mouse Ren2 transgene was detected in pancreas RNA extracts from all Ren2 rats examined but not in the pancreas of SD rats (Fig. 4A). There were increases in pancreas steady-state mRen2 transcript levels, adjusted for



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FIG. 2. Renin inhibition improves IRS-1 but not IRS-2 in Ren2 islet. A, Representative fluorescent images of pancreas tissue (I, Islet; E, exocrine tissue) IRS-1 (top). B, Quantification of converted signal intensities in average gray-scale intensities (bottom). C, Western blot analysis of total IRS-1. D and E, Representative fluorescent images of islet IRS-2 (bottom) and quantification (D) of converted signal intensities in average gray-scale intensities (top). *, P ⬍ 0.05 compared with SD-C; **, P ⬍ 0.05 when Ren2-A is compared with Ren2-C. Scale bar, 50 ␮m.

GAPDH level, when aliskiren-treated Ren2 were compared with Ren2 controls (0.31 ⫾ 0.2 vs. 0.14 ⫾ 0.1 arbitrary units, respectively). Analysis of fluorescent images and quantification demonstrated Ang II levels were higher in Ren2-C in both islets and exocrine tissue (62.7 ⫾ 1.5 and 32.4 ⫾ 1.6 average grayscale intensities, respectively) compared with SD-C (43.9 ⫾ 1.3 and 26.8 ⫾ 3.7 average gray-scale intensities, respectively; each P ⬍ 0.05) (Fig. 4, B and C), and levels of Ang II in both islets and exocrine pancreatic tissue were significantly reduced with aliskiren treatment (34.4 ⫾ 6.5 and 20.2 ⫾ 1.5 average gray-scale intensities, respectively; each P ⬍ 0.05). Pancreas oxidative stress

We used 3-nitrotryosine staining as a marker of peroxynitrite formation to ascertain islet and exocrine pancreas oxidative stress. There were higher levels of 3-nitrotyrosine immunostaining in the Ren2 islet and exocrine pancreas (94.3 ⫾ 12.1 and 79.5 ⫾ 5.2 average gray-scale intensities, respectively) compared with SD controls (39.9 ⫾ 4.4 and 61.3 ⫾ 3.0 average gray-scale intensities, respectively; each P ⬍ 0.05) (Fig. 5, A and B) improved by aliskiren treatment (56.5 ⫾ 7.5 and 60.7 ⫾ 8.0 average gray-scale intensities, respectively; each P ⬍ 0.05).

Pancreas NADPH oxidase activity and subunits

There were increases total enzyme activity in the Ren2 (1.29 ⫾ 0.21 mOD/min䡠mg) when normalized to SD control improved with aliskiren treatment in the Ren2 (0.96 ⫾ 0.16 mOD/min䡠mg). There were similar increases in immunostaining of p47phox in the Ren2 islet and exocrine pancreas (138.8 ⫾ 7.3 and 91.2 ⫾ 12.4 average gray-scale intensities, respectively) when compared with SD controls (66.4 ⫾ 15.1 and 39.0 ⫾ 3.9 average gray-scale intensities, respectively; each P ⬍ 0.05) (Fig. 6B), and this immunostaining intensity was significantly decreased with aliskiren treatment (90.0 ⫾ 4.6 and 53.8 ⫾ 3.4 average gray-scale intensities, respectively; each P ⬍ 0.05). Similarly, there were increases in Rac1 immunostaining in the Ren2 islet and exocrine pancreas (64.2 ⫾ 7.8 and 105.2 ⫾ 13.7 average gray-scale intensities, respectively) when compared with SD controls (39.6 ⫾ 5.2 and 26.6 ⫾ 3.1 average gray-scale intensities, respectively; each P ⬍ 0.05) (Fig. 6C), and both islet and exocrine Rac1 immunostaining decreased with aliskiren treatment (50.2 ⫾ 1.2 and 32.0 ⫾ 3.3 average gray-scale intensities, respectively; P ⬍ 0.05). Structural remodeling changes: light microscopic changes

VVG staining of pancreatic tissue was used to evaluate the effect of aliskiren on pancreatic perivascular and interlobular

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FIG. 3. Renin inhibition improves total and phosphorylated islet Akt. A, Representative fluorescent images of pancreas tissue (I, Islet; E, exocrine tissue) total Akt (top). B, Quantification of converted signal intensities in average gray-scale intensities (bottom). C, Western blot analysis of serine (Ser473) phosphorylated Akt. D and E, Representative fluorescent images of threonine (Thr) 308 and Ser473 phosphorylated Akt (bottom) and quantification (D) of converted signal intensities in average gray-scale intensities (top). *, P ⬍ 0.05 compared with SD-C; **, P ⬍ 0.05 when Ren2-A is compared with Ren2-C. Scale bar, 50 ␮m.

fibrosis. In Ren2-C there were increases in interlobular fibrosis only in the exocrine pancreas, which were associated with adventitial fibrosis of (52.2 ⫾ 2.0 percent) compared with SD-C (30.4 ⫾ 1.3 percent, P ⬍ 0.05). Furthermore, there were substantial reductions in fibrosis in aliskiren-treated Ren2A rats (35.2 ⫾ 1.8%, P ⬍ 0.05) (Fig. 7A). Conversely, there were decreases in the lumen size of Ren2-C (30.0 ⫾ 1.6%) compared with SD-C (44.5 ⫾ 1.3%, P ⬍ 0.05), which were increased in the aliskiren-treated Ren2-A (39.5 ⫾ 1.8, P ⬍ 0.05). Ultrastructural analysis of pancreas islet remodeling via TEM

Because periislet/islet exocrine interface (IEI) fibrosis is not evident on light microscopic staining, we decided to examine this region for ultrastructural changes of fibrosis by TEM. Accordingly, we evaluated the IEI to examine for the presence of very early cellular changes and extracellular matrix deposition of collagen (i.e. early IEI fibrosis). In the SD pancreas, there was an abrupt transition from the islet region

to the exocrine pancreas at a magnification ⫻10,000 (Fig. 7C, top left panel). There were observational findings of a thickening of the IEI at ⫻6,000 in the Ren2 (Fig 7C, top right panel), which suggests extracellular matrix deposition. In the Ren2 pancreas, there was endoplasmic reticulum (ER) compacting of the adjacent exocrine acinar cells, and at higher magnification (⫻25,000), fibrillar-banded collagen was noted (Fig. 1C, bottom right panel). This very early IEI fibrosis in the placebo-treated Ren2 pancreas was not present in any of the SD-C-, SD-A-, or Ren2-A-treated animals. There were no notable observational differences between the SD-C, SD-A, Ren2-C, and Ren2-A rats regarding intraislet ultrastructural remodeling including ␤-cell apoptosis and ␤-cell ultrastructural organelles (insulin secretory granules, Golgi apparatus, and nuclear contents) identified by TEM. Interestingly, in the placebo-treated Ren2 islets, the ␤-cell mitochondria displayed aggregation or clumping of the mitochondrial network in those ␤-cells that were depleted of their insulin secretory granules. Furthermore, in these same ␤-cells, there was an increased presence of ER by TEM. These



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FIG. 4. Ang II in the pancreas of the Ren2. A, RT-PCR was performed on total RNA extracts from pancreas to detect transcription of the mouse Ren2 gene. The upper row of bands in the inset is representative of the average level of mouse Ren2 transcript, and the lower row is the respective sample rat GAPDH transcripts. Below is the densitometry analysis of the transcripts normalized to GAPDH. B, Representative images of pancreas tissue (I, Islet; E, exocrine tissue) Ang II. C and D, Quantification of converted signal intensities in average gray-scale intensities in islet and exocrine pancreas. *, P ⬍ 0.05 when compared with SD-C; **, P ⬍ 0.05 when Ren2-A is compared with Ren2-C. Scale bar, 50 ␮m.

mitochondrial and ER changes within the ␤-cell were not observed in the SD-C, SD-A, or Ren2-A islets. These TEM observations may help to explain the increased mitochondrial staining intensity by immunostaining presented later. Mitochondrial quantification

Total mitochondria content was determined by measuring citrate synthase activity, an enzyme used to quantify mitochondrial content. There were increases in the Ren2 (52.2 ⫾ 2.6 mmol/min䡠mg) citrate synthase activity when compared with SD controls (41.8 ⫾ 3.6 mmol/min䡠mg, P ⫽ 0.06) (Fig. 8A), and mitochondrial content decreased with aliskiren treatment (38.1 ⫾ 2.6 mmol/min䡠mg, P ⬍ 0.05). There were no changes observed in ␤-HAD activity, a marker for fatty acid oxidation and mitochondrial source of oxidative stress (Fig. 8B). Thus, the increased mitochondrial content did not

translate into increased mitochondrial generation of ROS. Analysis of ultrastructural (Fig. 8C) and fluorescent images revealed the relative number of mitochondria in both islet and exocrine tissue of Ren2 (50.0 ⫾ 13.3 and 28.5 ⫾ 1.9 relative numbers, respectively) were also greater compared with SD controls (21.3 ⫾ 10.1 and 20.8 ⫾ 1.9 relative numbers, respectively) (Fig. 8, D–G). The numbers of mitochondria in Ren2 pancreas decreased substantially after in vivo aliskiren treatment (20.0 ⫾ 4.6 and 22.5 ⫾ 2.2 relative numbers, respectively). Discussion

In this investigation, we observed that 7- to 9-wk-old male transgenic Ren2 rats are insulin resistant in comparison with age-matched SD littermates. This observation is consistent with previous studies (9, 18). In concert with the insulin

A SD-C

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FIG. 5. Renin inhibition improves pancreas 3-nitrotyrosine immunostaining, as a marker for peroxyntitrite formation. A, Representative images of pancreas islet (I) (top) with quantification of converted signal intensities in average gray-scale intensities (bottom). Scale bar, 50 ␮m. B, Representative images of exocrine pancreas (top) with quantification of converted signal intensities in average gray-scale intensities (bottom). Scale bar, 20 ␮m. *, P ⬍ 0.05 when compared with SD-C **, P ⬍ 0.05, when Ren2-A is compared with Ren2-C.

SD-A

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p47phox

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FIG. 6. Renin inhibition reduces NADPH oxidase subunits. A, Representative images of immunohistochemical staining of NADPH oxidase subunits p47phox and Rac1 (I, Islet; E, exocrine tissue). B, Gray-scale intensity measures of A in the pancreas islet. C, Gray-scale intensity measures of A in the exocrine pancreas. *, P ⬍ 0.05 compared with SD-C; **, P ⬍ 0.05 when Ren2-A is compared with Ren2-C. Scale bar, 50 ␮m.

resistance/hyperinsulinemia, there was increased islet levels of insulin, renin transgene expression, and Ang II immunostaining. In parallel with increased pancreas Ang II immunostaining, there were increases in NADPH oxidase activity

A SD-C

SD-A

and nitrotyrosine immunostaining in the islets of Ren2 rats. These observations are consistent with prior reports suggesting a role of increased tissue RAS contributing to enhanced tissue oxidative stress in animal models of insulin

C X X

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FIG. 7. A, Perivascular fibrosis in the Ren2 pancreas. Representative images of light microscopy of structural changes consistent with perivascular fibrosis using VVG staining. B, Graph depicting the percent areas of perivascular fibrosis in the Ren2. *, P ⬍ 0.05 when compared with SD-C; **, P ⬍ 0.05 when Ren2-A is compared with Ren2-C. C, Ultrastructural evaluation of IEI fibrosis within the pancreas using TEM. Top left panel depicts the normal appearance of the IEI (black arrows) between the endocrine islet and exocrine region of the pancreas in the SD control. Box arrow (white) indicates zymogen granules in the exocrine pancreas compared with the much smaller (electron dense) islet secretory granules (X). Magnification, ⫻10,000. Scale bar, 2 ␮m. Top right panel demonstrates the IEI region (open arrow) is somewhat expanded in the Ren2-C compared with the higher magnification of ⫻10,000 in the SD-C. The areas of early fibrosis (black arrow) are even more evident at higher magnification in bottom right panel. In addition to early fibrotic changes, the Ren2-C demonstrates ER compacting in the exocrine pancreas (white star). Magnification, ⫻6000. Scale bar, 1 ␮m. Bottom left panel demonstrates disordered collagen fibrils (X) in close association with IEI and increased rER (rough endoplasmic reticulum). Magnification, ⫻25,000. Scale bar, 200 nm. Bottom right panel displays both orderly longitudinal and cross-section fibrillar-banded collagen (white X) in the Ren2 control. The arrow depicts the IEI region between the endocrine and exocrine pancreas not found in the SD-C. Note the liberation of zymogen granule contents (black X) into the IEI region. Magnification, ⫻25,000. Scale bar, 200 nm.



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FIG. 8. Pancreas mitochondria in the Ren2. A, Citrate synthase activity as a marker for mitochondrial content in pancreas tissue. B, ␤-HAD activity, a marker for fatty acid oxidation and mitochondrial source of oxidative stress in the pancreas. C, Representative image of the Ren2-C ␤-cells within the center of the islet on TEM. Note the increased aggregation and clumping of the mitochondrial network within the ␤-cells (arrows, surrounded by dashed white lines). Also note the lack of insulin secretory granules (open arrows) in these areas of increased mitochondria. This type of image is prevalent within the islets of the Ren2 model and were not noted within the islets of the SD control model. Magnification, ⫻4000. Scale bar, 2 ␮m. D, Representative fluorescent images of pancreas incubated with anticomplex IV subunit 1. E, Fluorescent images from C were merged with the transmitted images showing the islet structure I, Islet; E, exocrine tissue. Quantification of relative number of mitochondria in both islet (F) and exocrine pancreas (G). *, P ⬍ 0.05 compared with SD-C; #, P ⫽ 0.06 when compared with SD-C; **, P ⬍ 0.05 when Ren2-A is compared with Ren2-C. Scale bar, 50 ␮m.

resistance and type 2 diabetes (2– 8, 18, 22, 23). Enhanced Ang II-mediated activation of NADPH oxidase activity (11) was further evidenced by the finding of increases in p47phox and Rac1, critical subunits for activation of this enzyme in Ren2 islet tissue. Increases in islet Ang II immunostaining were accompanied by decreases in immunostaining for IRS-1 and -2 and Akt. In vivo, treatment with aliskerin was associated with contemporaneous ex vivo decreases in islet Ang II and increased total IRS-1 and Akt phosphorylation at both the Ser473 and Thr308 moieties. These observations are consistent with increased insulin metabolic signaling after renin inhibition. Our data support the notion that prorenin and renin are synthesized in the pancreas (3– 6). Renin catalyzes the initial rate-limiting step of the RAS, and that angiotensinogen is its only naturally occurring substrate, allows for highly specific inhibition of this system and additionally blocks renin from binding with the renin/prorenin receptors in pancreas (3–5) and other tissues (24, 28, 29). Aliskiren is the first of a new class of nonpeptide renin inhibitors, which has selective high-affinity binding to only human and mouse renin (24). Accordingly, we used a rodent model of insulin resistance/ type 2 diabetes that overexpresses the murine renin gene (9 –15). This allowed for the interruption of pancreatic RAS at the initial rate-limiting step of renin activation. Pancreatic islets are highly vulnerable to oxidative stress because islets have a low intrinsic antioxidant capacity (17,

19). In this regard, increases in NADPH oxidase without changes in ␤-HAD, an indicator of mitochondrial fatty acid oxidation, suggest that NADPH oxidase is the sources of increased pancreatic ROS in the Ren2 rat. Increases in nitrotyrosine in parallel with Ang II and NADPH oxidase activity suggests that an Ang II mediates the generation of ROS via increases in NADPH oxidase in the Ren2 pancreas. The observations in this investigation are consistent with the idea that oxidative stress may play an important role in islet ␤-cell injury that occurs in the early stages of the evolution of type 2 diabetes (17–19, 31–34). Our observations are also consistent with the notion that RAS blockade may reduce pancreatic ␤-cell damage (29, 33). ␤-Cells express both pro-renin and AT1R genes (3), and overexpression of the AT1R has been shown to decrease first-phase insulin secretion in isolated islets from the db/db mouse (23). When these db/db mice were treated with an AT1R blocker, they demonstrated a reduction in hyperglycemia, an improvement of their impaired glucose tolerance, and a delay in onset of diabetes (23). Similarly, in the Zucker rat (6), treatment with an ACE inhibitor or an AT1R blocker has been shown to decrease islet nitotyrosine levels and fibrosis, and restore the glucose-induced first phase insulin secretion. Thus, the observations in the current and prior investigations underscore the importance of an activated tissue RAS in promotion of ␤-cell dysfunction. Islet structural changes in the Ren2 pancreas included

5652 Endocrinology, November 2008, 149(11):5643–5653

increased numbers of structurally abnormal mitochondria, exocrine perivascular fibrosis, and increased fibrosis at the islet exocrine pancreas interface. Most of these functional and structural abnormalities observed in the ex vivo pancreatic islet were significantly improved by 3 wk of in vivo treatment with aliskerin. Improvements in pancreatic structural abnormalities occurred in conjunction with reductions in NADPH oxidase activity and nitrotyrosine content. Accordingly, these data indicate that renin inhibition offers a potential new strategy to preserve the structural and functional integrity islets by reducing NADPH oxidase activity/ROS generation. RAS activation of NADPH oxidase activity/subunit expression and increases in nitrotyrosine in the exocrine portion of the Ren2 pancreas may help explain the increased fibrosis in the islet-exocrine interface region and perivascular fibrosis in the exocrine region of the Ren2 pancreas observed in this study. This is also consistent with the increased renin expression found in all tissues of the Ren2 rat (9), and the increased Ang II levels that were also observed in the pancreatic exocrine tissue. In the exocrine pancreas, angiotensinogen, AT1, and AT2 receptors are localized in the pancreatic ducts, blood vessels, and acinar cells (4, 5, 35, 36). This local RAS system appears to be involved in the regulation of the pancreatic microcirculation, acinar enzyme secretion, and pancreatic pericyte/stellate cell function (35–38). Our current observations that renin blockade reduces oxidative stress, with concurrent reductions in NADPH oxidase activity/ subunit expression, provide a mechanism for the prior observations that treatment with an AT1R blocker and ACE inhibitor attenuate pancreatic exocrine inflammation and fibrosis in an experimental model of pancreatitis (37, 38). The increased numbers of structurally abnormal mitochondria and the more prominent endoplasmic reticulum in the islets of the Ren2 rats may represent further manifestations of ␤-cell dysfunction and production of oxidative stress. The increased ROS generated by increased activation of NADPH oxidase may represent a feed-forward activation of mitochondrial production of ROS via citrate synthase and the electron transport chain (19, 39, 40). Krauss et al. (39) demonstrated that superoxide can activate uncoupling protein-2. Activation of uncoupling protein-2 diverts energy away from ATP synthesis, increases mitochondrial uncoupling generation of ROS, and decreases the ATP to ADP ratio, resulting in less efficient glucose modulation of insulin secretion (39, 40). Superoxide radicals also lead to irreversible decreases in the level of transcription factor pancreatic duodenal homeobox-1 (19), which is critical for ␤-cell neogenesis. Increased superoxide production resulting from both NADPH oxidase and mitochondrial generation may lead to increased phosphorylation of IRS-1 on Ser307, which targets IRS-1 for proteolytic degradation and reduced total IRS-1 as well as engagement with phosphatidylinositol 3-kinase and downstream Akt activation as seen in the Ren2 islet. The enhanced oxidant stress in the presence of increased islet RAS activity may have accounted for the increased prominence of the ER in islets of Ren2 rat. Indeed, oxidative stress may enhance endoplasmic stress (41), and this further contributes to ␤-cell dysfunction, in part, by increasing IRS-1 Ser phosphorylation and targeted degradation of IRS-1 (30). Interestingly, renin inhibition corrected the islet mitochon-


drial abnormalities, normalized ROS levels, and increased IRS-1 as well as Akt phosphorylation in the Ren2 islet. Indeed, these effects on mitochondria in conjunction with diminished IRS-1 were abrogated by renin inhibition, further highlighting the importance of the local islet RAS in promoting ␤-cell dysfunction. Acknowledgments Male transgenic Ren2 rats and male SD controls were kindly provided by Wake Forest University School of Medicine (Winston-Salem, NC) through the Transgenic Core Facility supported in part by National Institutes of Health Grant HL-51952. The authors acknowledge the electron microscope core facility for their help and preparation of transmission electron micrographs. Received January 16, 2008. Accepted July 15, 2008. Address all correspondence and requests for reprints to: James R. Sowers, M.D., A.S.C.I., A.P.S., F.A.C.P., Thomas W. and Joan F. Burns Missouri Chair in Diabetes, Professor of Medicine, Physiology, and Pharmacology, D109 HSC Diabetes Center, One Hospital Drive, Columbia, Missouri 65212. E-mail: [email protected]. This work was supported by National Institutes of Health Grants R01 HL73101-01A1 (to J.R.S.) and HL-51952 (to C.M.F.); Veterans Affairs Merit System (0018) (to J.R.S.) and VISN 15 (to A.W.-C.); Missouri Kidney Program (to A.W.-C.); and Novartis Pharmaceuticals (to J.R.S. and A.W.-C.). Disclosure Statement: J.R.S. and A.W.-C. report having received grant funding from Novartis; J.R.S. reports being on advisory board for Novartis, Forest, and Merck; and J.H., M.R.H., V.G.D., R.S., S.D.S., P.K., and C.M.F. have nothing to declare.

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