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Hypertension. 1997;29:728-735

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(Hypertension. 1997;29:728-735.)
© 1997 American Heart Association, Inc.

Articles

Characteristics of Diabetes, Blood Pressure, and Cardiac and Renal Complications in Otsuka Long-Evans Tokushima Fatty Rats

Keiko Yagi; Shokei Kim; Hideki Wanibuchi; Tatsuya Yamashita; Yoshitaka Yamamura; Hiroshi Iwao

the Department of Pharmacology (K.Y., S.K., H.I.) and First Department of Pathology (H.W.), Osaka City University Medical School, and the Second Tokushima Institute of New Drug Research (T.Y., Y.Y.), Otsuka Pharmaceutical Co, Ltd, Tokushima, Japan.

Correspondence to Shokei Kim, MD, Department of Pharmacology, Osaka City University Medical School, 1-4-54 Asahimachi, Abeno, Osaka 545, Japan.


*    Abstract
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 *Abstract
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To characterize the molecular mechanism of cardiac and renal complications in non–insulin-dependent diabetes mellitus (NIDDM), we examined the gene expression of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a new animal model for human NIDDM, at the ages of 14 weeks (prediabetic stage), 30 weeks (NIDDM stage), and 54 weeks (IDDM stage). Tissue mRNA levels were measured by Northern blot analysis. In 14-week-old OLETF rats, cardiac mRNAs for transforming growth factor-ß1 (TGF-ß1) and extracellular matrix, including collagen types I, III, and IV and laminin, were significantly increased compared with control rats (Long-Evans Tokushima Otsuka rats). Cardiac ß-myosin heavy chain (MHC) mRNA of OLETF was increased at 30 and 54 weeks of age, whereas {alpha}-MHC mRNA of OLETF was inversely decreased at 54 weeks. Marked perivascular fibrosis was seen in the hearts of OLETF rats from 30 weeks of age. In the kidney of OLETF rats, glomerular TGF-ß1 expression was temporally increased at 30 weeks of age, followed by glomerulosclerosis characterized by mesangial proliferation, thickening of the basement membrane, and nodular lesions. Blood pressure of OLETF rats remained higher than that of control rats from the prediabetic stage to the IDDM stage. Thus, in OLETF rats, cardiac fibrosis–related gene expressions were already enhanced at the prediabetic stage, which supports the involvement of these gene expressions in cardiac perivascular fibrosis. The antithetical change in ß- and {alpha}-MHC expressions seems to participate in the decreased cardiac contractility seen in diabetes. Furthermore, TGF-ß1 may also contribute to glomerulosclerosis of OLETF rats. OLETF rats seem to be a useful model to study the mechanism of hypertension and cardiac and renal complications in NIDDM.


Key Words: diabetes mellitus • heart • nephropathy, diabetic • transforming growth factor • blood pressure


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Cardiovascular and renal complications are the major causes of mortality in DM.1 2 However, the detailed mechanism and characteristics of these diabetic complications remain to be elucidated, mainly because of the scarcity of a suitable animal model with diabetic complications similar to human diabetes. Accumulating evidence on cardiac performance–related gene expressions shows that the altered gene expressions of cardiac contractile proteins, including MHC and {alpha}-actin, and sarcoplasmic reticulum Ca2+-ATPase participate in the development of cardiac dysfunction or heart failure.3 4 5 Furthermore, the increased gene expression of extracellular matrix components, such as collagen, and TGF-ß1 causes cardiac fibrosis and increases cardiac stiffness, leading to cardiac diastolic dysfunction.6 7 8 9 However, there is no report concerning these cardiac gene expressions in diabetic cardiomyopathy.

In 1992, a new animal model of NIDDM, the OLETF rat, was established from a spontaneously diabetic Long-Evans rat from Charles River Canada (St Constant, Quebec, Canada) by selective mating.10 OLETF rats are characterized by (1) late onset of hyperglycemia (after {approx}20 weeks of age), (2) a mild and chronic course of DM, (3) the conversion of DM from NIDDM to IDDM after about 40 weeks of age, (4) mild obesity, and (5) inheritance by males.10 Interestingly, the pathological features of glomerulosclerosis observed in OLETF rats are similar to those in human DM. However, it is unknown whether OLETF rats display cardiac complications and hypertension. Moreover, the mechanism of nephropathy in OLETF rats remains to be determined.

In the present study, to characterize cardiac complications in OLETF rats, we examined not only pathological features of the heart but also the above-mentioned cardiac gene expressions of OLETF rats at the stages of prediabetes (14 weeks), NIDDM (30 weeks), and IDDM (54 weeks). In addition, we also studied the possible contribution of TGF-ß1 to nephropathy of OLETF rats.


*    Methods
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 *Methods
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Animals
All procedures were in accordance with institutional guidelines for animal research. LETO were used as the genetic control of OLETF. Male OLETF and LETO rats were maintained at the Tokushima Research Institute (Otsuka Pharmaceutical). All rats were kept at the Specific Pathogen-Free facility under controlled temperature (23±2°C) and humidity (55±5%) with a 12-hour light and dark cycle. They were fed standard laboratory chow (MF, Oriental Kobo) and given tap water ad libitum.

Experimental Protocol
Body weight, blood pressure, plasma glucose, and urinary protein excretion of OLETF and LETO rats were monitored periodically from the age of 5 to 54 weeks. For measurement of urinary protein excretion, rats were housed individually in metabolic cages, and urine was collected for 24 hours.

OLETF and LETO rats at the ages of 14 weeks (prediabetic phase), 30 weeks (NIDDM phase), and 54 weeks (IDDM phase) were decapitated, trunk blood was collected for measurement of PRC, and the heart and kidneys were rapidly excised. The left ventricle was immediately separated from the right ventricle and the atria, weighed, frozen in liquid nitrogen, and stored at -80°C until the extraction of total tissue RNA. The renal cortex was separated from the left kidney and one half of the right kidney from individual rats, and glomeruli were isolated by the sieving method of MacKay et al.11 The isolated glomeruli were immediately frozen in liquid nitrogen and stored at -80°C until the extraction of total RNA. The remaining half of the right kidney was used for immunohistochemistry of TGF-ß1, as described below.

Northern Blot Analysis of mRNAs From Heart and Glomeruli
Total RNA extraction and the subsequent Northern blot hybridization were carried out as previously described in detail.12 13 In brief, total RNA was extracted from the left ventricle and the isolated glomeruli of OLETF and LETO rats according to the guanidinium thiocyanate–phenol-chloroform method. Total RNA denatured by glyoxal and dimethyl sulfoxide was electrophoresed on 1% agarose gel and transferred to a nylon membrane (Gene Screen Plus, EI du Pont de Nemours & Co, NEN Products). The 28S and 18S ribosomal RNAs in gels were stained with ethidium bromide to demonstrate the integrity of applied RNA and to verify that the same amounts of RNA were applied to each lane. For LV mRNAs for {alpha}- and ß-MHC and skeletal and cardiac {alpha}-actin, Northern blot hybridization was performed with specific oligonucleotide probes labeled with [{gamma}-32P]ATP (6000 Ci/mmol, Amersham Co) at the 5' end with T4 polynucleotide kinase. The method of Northern blot analysis with oligonucleotide probes, including the prehybridization, hybridization, and washing of the membranes and the subsequent autoradiography, has been described in detail.13 For other mRNAs, Northern blot analysis was carried out with cDNA probes labeled with [32P]dCTP (specific activity, 3000 Ci/mmol, New England Nuclear) by the random primer extension method. The conditions of prehybridization, hybridization, washing of the membranes, and the subsequent autoradiography were previously described in detail.12 The autoradiographic density of mRNA bands was measured by a Macintosh LC-III computer with an optical scanner (EPSON GT-8000, Seoko) using the public domain NIH Image program. In all RNA samples, the hybridization signals of specific mRNAs were normalized for those of GAPDH mRNA to correct for differences in RNA loading and/or transfer.

After autoradiography, the membranes were boiled in 0.1xSSC containing 1% sodium dodecyl sulfate for 30 minutes to strip off the hybridized oligonucleotide or cDNA probe and were then rehybridized with other oligonucleotide or cDNA probes.12 13

cDNA and Oligonucleotide Probes
For measurement of MHC and {alpha}-actin isoform mRNAs, we used synthetic oligonucleotide probes complementary to the unique 3' untranslated regions of these mRNAs, as previously described,13 because the 3' untranslated regions are not closely conserved between {alpha}- and ß-MHC mRNAs14 and between skeletal and cardiac {alpha}-actin mRNAs.15 16 The sequences of oligonucleotide probes used were as follows: {alpha}-MHC, 5'-TTGTGGGATAGCAACAGCGA-3'; ß-MHC, 5'-GTCTCAGGGCTTCACAGG-3'; skeletal {alpha}-actin, 5'-GCAACCATAGCACGATGGTC-3'; and cardiac {alpha}-actin, 5'-TGCACGTGTGTAAACAAACT-3'.

cDNA probes used were as follows: rat TGF-ß1 cDNA (1.0-kb HindIII–Xba I fragment)17 ; rat {alpha}1(I) collagen (1.3-kb Pst I–BamHI fragment)18 ; mouse {alpha}1(III) collagen (1.8-kb EcoRI-EcoRI fragment)19 ; mouse {alpha}1(IV) collagen (0.83-kb Aba I–Pst I fragment)20 ; laminin B2 chain (1.7 kb Xba I/EcoRI fragment)21 ; rat ANP cDNA (0.825-kb fragment)13 ; rabbit sarcoplasmic reticulum Ca2+-ATPase (2.0-kb BamHI fragment)22 ; and GAPDH (1.3-kb Pst I–Pst I fragment).23

Immunohistochemistry of TGF-ß1
Immunohistochemistry of TGF-ß1 was carried out as described.24 Kidney obtained from 14-, 30-, and 54-week-old OLETF and LETO rats was fixed in methyl Carnoy's solution (60% methanol, 30% chloroform, 10% acetic acid), embedded in paraffin, and cut into 4-µm-thick sections. The sections were deparaffinized in xylene and a graded series of ethanols. Immunostaining was carried out by the streptavidin-biotin immunoperoxidase method (DACO LSAB kit, DAKO Corp). The sections were immersed in 3% hydrogen peroxide to quench the endogenous peroxidase activity and then incubated with PBS containing 1% BSA to reduce the nonspecific background staining. The sections were rinsed with PBS and incubated with specific anti–TGF-ß1 chicken IgG (AB-101-NA, R&D Systems Inc), diluted 1:100, at 4°C for 24 hours. After being washed with PBS, the sections were incubated with biotinylated goat anti-chicken immunoglobulin G, then washed with PBS, and further incubated with peroxidase-labeled streptavidin for 10 minutes. Sections were reacted with 3,3'-diaminobenzidine as the chromogen and counterstained with hematoxylin.

Semiquantitative analysis for glomerular expression of TGF-ß1 was performed and graded according to the method of Floege et al.25 Scores were as follows: 0, absent or very weak staining; 1+, staining involving 1% to 25% of the area of the glomerular tuft; 2+, staining involving 25% to 50% of the area of the glomerular tuft; 3+, staining involving 50% to 75% of the area of the glomerular tuft; and 4+, staining involving >75% of the area of the glomerular tuft.

Histological Examination
Fourteen-, 30-, and 60-week-old OLETF and LETO rats (n=5 to 7 per group at each age) were anesthetized with pentobarbital sodium. The heart and kidney were rapidly fixed by perfusion with 10% phosphate-buffered formalin from a catheter inserted into the left ventricle. The heart and kidney were rapidly removed, again fixed in the same formalin solution, and embedded in paraffin. Sections 4 µm thick were cut and stained with hematoxylin and eosin, Azan, or PAS.

Measurement of Blood Pressure
Systolic blood pressure was measured by the tail-cuff method (Riken Kaihatsu PS-8000).

Measurement of PRC
PRC was measured as the rate of Ang I generation from rat plasma angiotensinogen as described.26

Miscellaneous Measurements
For plasma glucose measurement, blood was taken from a tail vein and centrifuged for 20 minutes. Plasma glucose was determined by the glucose oxidase method with a Glucose-B Test kit (Wako Pure Industries, Ltd). Urinary protein excretions were measured with an A/G-B test (Wako Pure Industries, Ltd). Plasma insulin levels were determined with a radioimmunoassay kit (INCSTAR).

Statistics
Data are expressed as mean±SEM. The data were analyzed by two-way ANOVA, and the differences in the data between LETO and OLETF rats at each age and between different ages were determined by the least-squares means test (SuperANOVA, Abacus Concepts). Differences were considered statistically significant at a value of P<.05.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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Body Weight, Plasma Glucose Concentration, Urinary Protein Excretion, and Blood Pressure
As shown in Fig 1ADown, the body weight of OLETF rats was significantly greater than that of LETO from the age of 14 to 46 weeks but the body weights of OLETF gradually decreased after 38 weeks of age. There was no difference in body weight between these two strains at 54 weeks of age.



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  		Figure 1. Body weight (A), plasma glucose (B), urinary protein excretion (UprotV) (C), and blood pressure (D) of LETO and OLETF rats. Values are mean±SEM (n=14 for 5, 14, 22, and 30 weeks of age and n=7 for 38, 46, and 54 weeks of age). {dagger}P<.05, *P<.01 vs LETO rats.

Plasma glucose levels of OLETF rats increased significantly with aging and were significantly higher than those of LETO after 22 weeks of age (Fig 1BUp). In particular, the increase in plasma glucose of OLETF became much more prominent at >38 weeks of age, which was in good agreement with the previous report10 and can be explained by the conversion of DM from NIDDM to IDDM.10

As shown in Fig 1CUp, urinary protein excretion of 22-week-old OLETF rats was already significantly greater than that of LETO of the same age. As in plasma glucose, the increase became more prominent in OLETF rats at >38 weeks of age.

Blood pressure of OLETF rats was slightly increased with aging and was significantly higher than that of LETO throughout the age range of 14 to 54 weeks (Fig 1DUp).

LV Weight and Kidney Weight of OLETF Rats
As shown in Fig 2A and 2BDownDown, LV weight did not differ significantly between OLETF and LETO rats. However, the LV weight of 30-week-old OLETF rats tended to be larger than that of control LETO rats of the same age. Kidney weights of 30- and 54-week-old OLETF rats were 1.6- and 1.7-fold larger (P<.01), respectively, than in LETO of the same age (Fig 2CDown), and kidney weight corrected for body weight was 1.8-fold larger (P<.01) in 54-week-old OLETF than in LETO of the same age (Fig 2DDown).



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  		Figure 2. LV weight (A) and LV weight corrected for body weight (BW) (B), kidney weight (C), and kidney weight corrected for BW (D) of LETO and OLETF rats. Unfortunately, kidney weights of OLETF and LETO rats were not determined (ND) at 14 weeks of age. Values are mean±SEM (n=5 to 8). *P<.01 between LETO and OLETF rats.

LV Gene Expression Related to Cardiac Performance
Figs 3Down and 4Down indicate LV mRNA levels for contractile proteins, ANP, and sarcoplasmic reticulum Ca2+-ATPase. In LETO rats, ß-MHC mRNA levels increased with age and were 2.2-fold higher (P<.01) at 54 weeks than 14 weeks of age, whereas {alpha}-MHC mRNA levels decreased to 67% at 54 weeks compared with 14 weeks of age. ß-MHC mRNA levels of 30- and 54-week-old OLETF were 1.9-fold (P<.05) and 1.9-fold (P<.01) higher, respectively, than in LETO of the same age. {alpha}-MHC mRNA levels of 54-week-old OLETF were lower than in LETO of the same age (P<.01).



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  		Figure 3. Representative autoradiograms of Northern blot analysis of LV mRNAs for ß-MHC, {alpha}-MHC, skeletal and cardiac {alpha}-actin, ANP, Ca2+-ATPase, and GAPDH from 14- and 54-week-old LETO and OLETF rats. Each autoradiogram indicates mRNA band from different individual rat samples. Sizes of mRNA bands were 7.1 kb for ß-MHC, 7.1 kb for {alpha}-MHC, 1.7 kb for skeletal {alpha}-actin, 1.7 kb for cardiac {alpha}-actin, 1.4 kb for ANP, 4.5 kb for Ca2+-ATPase, and 1.4 kb for GAPDH.



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  		Figure 4. LV mRNA levels for ß-MHC, {alpha}-MHC, skeletal {alpha}-actin, cardiac {alpha}-actin, ANP, and Ca2+-ATPase from LETO ({square}) and OLETF ({blacksquare}) rats. In individual samples, each mRNA value was corrected for GAPDH mRNA value. The mean value of each mRNA in 14-week-old LETO rats is represented as 1. Each bar represents mean±SEM (n=5 to 8). {dagger}P<.05, *P<.01 between LETO and OLETF rats.

In contrast to the age-related changes in ß- and {alpha}-MHC expression of LETO rats, neither skeletal or cardiac {alpha}-actin mRNA levels of LETO were significantly altered with age, and there was no significant difference in these mRNA levels between LETO and OLETF rats at all ages examined. In LETO rats, ANP mRNA levels increased with age and were 3.3-fold higher at 54 weeks than at 14 weeks (P<.01). At 54 weeks of age, ANP mRNA levels of OLETF were 1.3-fold higher than in LETO rats (P<.05). Sarcoplasmic reticulum Ca2+-ATPase mRNA levels were not affected by aging and were not significantly different between the two strains of rats.

LV Gene Expression Related to Cardiac Fibrosis
As shown in Figs 5Down and 6,Down TGF-ß1 mRNA levels of 14, 30, and 54-week-old OLETF rats were 1.5-, 1.6-, and 1.3-fold (P<.01) higher, respectively, than in LETO rats. Furthermore, at 14 weeks of age, LV gene expression of collagen types I, III, and IV and laminin in OLETF rats was significantly enhanced compared with LETO rats of the same age. Collagen types III and IV and laminin mRNA levels of OLETF rats remained higher at 30 weeks of age than in LETO rats.



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  		Figure 5. Representative autoradiograms of Northern blot analysis of LV mRNAs for TGF-ß1, collagen types I, III, and IV, laminin, and GAPDH from 14- and 54-week-old LETO and OLETF rats. Each autoradiogram indicates mRNA band from different individual rat samples. Sizes of mRNA were 2.5 kb for TGF-ß1, 4.7 and 5.7 kb for collagen I, 5.9 kb for collagen III, 6.8 kb for collagen IV, 8.0 kb for laminin B2, and 1.4 kb for GAPDH.



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  		Figure 6. LV mRNA levels for TGF-ß1, collagen types I, III, and IV, and laminin from LETO and OLETF rats. In individual rats, each mRNA value was corrected for GAPDH mRNA value. Mean value of each mRNA in 14-week-old LETO rats is represented as 1. Each bar represents mean±SEM (n=5 to 8).

Histopathological Examination of the Heart
In 14-week-old OLETF rats, there was no significant perivascular fibrosis in the left ventricle compared with LETO of the same age. At the ages of 30 weeks (Fig 7Down) and 60 weeks, marked perivascular fibrosis was observed in the left ventricle of OLETF rats compared with age-matched LETO rats. Conversely, there was no significant fibrosis in cardiac interstitium of OLETF rats compared with LETO at all ages examined.



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  		Figure 7. Light microscopy of left ventricle from 30-week-old LETO (A) and OLETF (B) rats. Marked perivascular fibrosis was observed in 30-week-old OLETF rats compared with LETO rats of same age. However, there was no significant fibrosis in the cardiac interstitium. Azan stain. Bar=100 µm.

Glomerular TGF-ß1 and Collagen Expression
As shown in Fig 8Down, glomerular TGF-ß1 mRNA levels of 30-week-old OLETF were 2.0-fold higher than in LETO of the same age (P<.01). Although glomerular collagen type I mRNA was not detected in 14-week-old OLETF, 30- (Fig 8ADown) and 54-week-old OLETF rats showed the expression of collagen type I mRNA to significantly detectable amounts. In 14-, 30-, or 54-week-old LETO rats, collagen type I mRNA was not detected at all. There was no significant difference in glomerular collagen type III and type IV mRNA levels between LETO and OLETF rats at 14, 30 (Fig 8ADown), or 54 weeks of age.



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  		Figure 8. Autoradiograms of glomerular TGF-ß1, collagen types I, III, and IV, and GAPDH mRNAs from 30-week-old LETO and OLETF rats (A) and glomerular TGF-ß1 mRNA levels from 14-, 30-, and 54-week-old rats (B). A, Sizes of mRNA were 2.5 kb for TGF-ß1, 4.7 and 5.7 kb for collagen I, 5.9 kb for collagen III, 6.8 kb for collagen IV, and 1.4 kb for GAPDH. Isolated glomeruli were pooled from two individual rats. Each autoradiogram shows mRNA band from different glomerular samples. B, TGF-ß1 mRNA value in individual rats was corrected for GAPDH mRNA value. Mean value in 14-week-old LETO rats is represented as 1. Each bar represents mean±SEM (n=3 to 5).

Immunohistochemical results in Fig 9Down and the TableDown show that immunoreactive TGF-ß1 protein was only faintly detected in glomeruli of LETO rats at all ages examined. Conversely, marked staining for TGF-ß1 was seen in glomerular cells of 30-week-old OLETF, whose glomerular TGF-ß1 staining score was 9.8-fold greater than that of LETO of the same age (P<.01).



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  		Figure 9. Immunohistochemical staining of TGF-ß1 in kidney of 30-week-old LETO (A) and OLETF (B) rats. A, Faint staining for TGF-ß1 in glomeruli of LETO rats. B, In contrast, marked staining for TGF-ß1 in glomeruli of OLETF rats. Original magnification x200.


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  		Table 1. Glomerular Staining Score for TGF-ß1

Pathological Examination of the Kidney
PAS staining of the kidney from 60-week-old OLETF rats indicated diffuse glomerulosclerosis, characterized by mesangial proliferation, thickening of the basement membrane, and nodular lesions (Fig 10Down), which is in good agreement with a previous report by Kawano et al.10



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  		Figure 10. Light microscopy of kidney from 60-week-old LETO (A) and OLETF (B) rats. PAS stain. Bar=100 µm.

Plasma Insulin and PRC
Plasma immunoreactive insulin of 30-week-old OLETF was significantly higher than that of LETO of the same age (20.5±4.7 versus 8.8±1.2 ng/mL; P<.01; each, n=7). Conversely, at 54 weeks of age, plasma insulin of OLETF tended to be lower than that of LETO (4.4±2.3 versus 9.4±1.2 ng/mL; each, n=7).

There was no significant difference in PRC between OLETF and LETO rats at the age of 14 weeks (25±3 versus 18±3 ng Ang I·h-1·mL-1), 30 weeks (22±3 versus 23±2 ng Ang I·h-1·mL-1), or 54 weeks (19±3 versus 15±2 ng Ang I·h-1·mL-1).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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Previous clinical data show that myocardial dysfunction frequently occurs in diabetes even in the absence of coronary artery disease,27 28 thereby supporting the existence of primary diabetic cardiomyopathy.29 Furthermore, the abnormalities of cardiac function, including decreased cardiac contractility, are reported to occur in streptozotocin-induced diabetes in dogs30 and rats.31 32 However, the mechanism responsible for cardiomyopathy in DM, particularly in NIDDM, is poorly understood. Based on the clinical evidence that cardiovascular disease can precede the development of NIDDM, it has been hypothesized that cardiovascular disease and NIDDM spring from a "common soil" and have a common genetic antecedent.33 34 35 These findings show the importance of molecular study for the elucidation of pathogenesis and characteristics of cardiomyopathy in NIDDM, thereby leading us to investigate cardiac gene expressions of OLETF rats in detail.

Cardiac contractile proteins examined in the present study, including MHC and {alpha}-actin, play a crucial role in the modulation of cardiac performance.3 4 5 36 MHC consists of two isoforms, {alpha}- and ß-MHCs. {alpha}-MHC, which has higher Ca2+- and actin-activated ATPase activity, is associated with an increased shortening velocity of the cardiac fibers, whereas ß-MHC, which has lower ATPase activity, is associated with slower shortening velocity.3 4 5 Thus, the increase in the ratio of cardiac ß-MHC to {alpha}-MHC, which is due to increased ß-MHC, decreased {alpha}-MHC, or both, leads to decreased cardiac contractility. A previous report by Dillmann,37 who separated rat cardiac myosin protein into three components, V1 (a homodimer of {alpha}-MHC), V2 (a heterodimer of {alpha}-MHC and ß-MHC), and V3 (a homodimer of ß-MHC), by pyrophosphate PAGE, showed that streptozotocin-induced diabetes increases the relative percentage of cardiac V3 myosin protein in total myosin and decreases that of V1 myosin protein. However, the molecular mechanism for the diabetes-induced change in cardiac myosin protein remains unknown. In the present study, in LETO rats, cardiac ß-MHC gene expression increased with age, whereas {alpha}-MHC expression inversely decreased with age. We found higher cardiac ß-MHC mRNA levels in 30-week-old OLETF rats than in LETO of the same age and found not only higher ß-MHC mRNA but also lower {alpha}-MHC mRNA in 54-week-old OLETF rats than in LETO, thereby indicating that diabetes further accelerates the age-related increase in ß-MHC and the reciprocal decrease in {alpha}-MHC expression in the heart and that ß-MHC gene expression can be changed by diabetes in a more sensitive manner than {alpha}-MHC. Therefore, the decreased cardiac contractility seen in diabetes30 31 32 may be at least in part explained by the antithetical gene expressions of ß-MHC and {alpha}-MHC.

Hypertension is one of the major risk factors of cardiovascular diseases and is closely related to NIDDM.38 39 Therefore, comparison between NIDDM and genetic hypertension with respect to cardiac performance-related gene expression is important to elucidate the mechanism of development of cardiomyopathy. Interestingly, in contrast to our present data on the enhanced expression of cardiac ß-MHC in OLETF rats, we have found that SHR, the most popular model of human essential hypertension, were characterized by no change in cardiac ß-MHC gene expression even at the severely hypertensive phase.40 Furthermore, our previous reports40 41 show that cardiac skeletal {alpha}-actin and ANP gene expressions are significantly upregulated in SHR throughout the hypertensive phase, also different from our present results on OLETF rats. These findings provide evidence for the differential regulation of gene expression of cardiac contractile proteins and ANP between NIDDM and genetic hypertensive rats.

Previous pathological examination of hearts obtained at autopsy of diabetic patients indicated that significant cardiac fibrosis was found in diabetic patients.42 However, little information is available on the mechanism of diabetic cardiac fibrosis, primarily because of the scarcity of a suitable animal model of diabetic cardiomyopathy. Of note are the observations that OLETF displayed prominent perivascular fibrosis. Therefore, we examined cardiac gene expression of extracellular matrix components, including collagen types I and III (the main interstitial collagen) and collagen type IV and laminin (the main basement membrane components). Moreover, we examined the expression of TGF-ß1, a growth factor that is involved in tissue fibrosis by stimulating extracellular matrix synthesis.6 43 Notably, cardiac gene expression of extracellular matrix components in OLETF rats was already significantly enhanced at the prediabetic stage (14 weeks), which suggested that these increased gene expressions may participate in the onset of cardiac fibrosis of OLETF rats. Furthermore, cardiac TGF-ß1 mRNA levels of OLETF rats remained increased from the prediabetic stage to the IDDM stage (54 weeks), which is in contrast to no alteration in cardiac TGF-ß1 in SHR.40 44 Thus, genetic factors may partially contribute to cardiac perivascular fibrosis in OLETF rats. However, further study is needed to elucidate the real involvement of TGF-ß1 in perivascular fibrosis in OLETF rats, because the present study did not allow us to determine the protein levels of TGF-ß1.

A previous report10 and our present study show that renal pathological features of OLETF rats are similar to those of human diabetes. However, no information is available for the mechanism of renal complications in OLETF rats. Accumulating evidence supports the hypothesis that TGF-ß1 is involved in glomerulosclerosis in various renal diseases12 24 43 45 by increasing the deposition of glomerular extracellular matrix. The increased glomerular expression of TGF-ß1 has been reported in streptozotocin-induced diabetes in rats46 47 and type I human diabetes,46 suggesting the possible contribution of TGF-ß1 in diabetic nephropathy. Therefore, in this study, we examined the gene expression of TGF-ß1 not only in the heart but also in the kidney of OLETF rats. Unlike the heart, glomerular TGF-ß1 expression of OLETF rats was not altered at the prediabetic stage (14 weeks) but was temporally increased at the stage of NIDDM preceding glomerulosclerosis. Thus, it is possible that TGF-ß1 may be partially responsible for the development of glomerulosclerosis in OLETF rats. In addition, the transient increase in glomerular TGF-ß1 differed from the continuous increase in cardiac TGF-ß1, indicating organ-specific regulation of TGF-ß1 in OLETF rats.

Notably, even at the stage of prediabetes, the blood pressure of OLETF rats was slightly but significantly higher than that of LETO rats. However, the plasma concentrations of renin, the limiting factor of the renin-angiotensin system, were not different between OLETF and LETO rats throughout all ages examined. Thus, the increased blood pressure of OLETF seems to be due to genetic factors rather than hyperglycemia or circulating renin-angiotensin system. As shown in Fig 2AUp, LV weights of 30-week-old OLETF rats tended to be larger than those of LETO rats of the same age. Furthermore, in another experiment, we have also compared 46-week-old OLETF and LETO rats and found that LV weights of OLETF rats at this age were significantly greater than in LETO rats (S.K. et al, unpublished data), thereby supporting the proposition that OLETF rats develop LV hypertrophy. The decreased LV weight in OLETF at 54 weeks may be explained by the metabolic disturbance accompanying severe IDDM.

In conclusion, already at the prediabetic stage, cardiac gene expression of TGF-ß1 and extracellular matrix components in OLETF rats was increased, which may be responsible for cardiac perivascular fibrosis in OLETF rats. Thus, our present study supports the "common soil" hypothesis33 34 35 that NIDDM and cardiomyopathy have a common genetic antecedent rather than one being a complication of the other. The increased TGF-ß1 gene expression may also participate in the development of nephropathy in OLETF rats. Furthermore, higher blood pressure in OLETF rats compared with LETO rats may be attributable to genetic factors. We propose that OLETF rats are a useful animal model to study the mechanism of cardiac and renal complications and hypertension in NIDDM.


*    Selected Abbreviations and Acronyms
 
Ang I = angiotensin I
ANP = atrial natriuretic polypeptide
DM = diabetes mellitus
IDDM = insulin-dependent diabetes mellitus
LETO = Long-Evans Tokushima Otsuka (rats)
MHC = myosin heavy chain
NIDDM = non–insulin-dependent diabetes mellitus
OLETF = Otsuka Long-Evans Tokushima Fatty (rats)
PRC = plasma renin concentration
SHR = spontaneously hypertensive rat(s)
TGF-ß1 = transforming growth factor-ß1


*    Acknowledgments
 
This work was supported in part by a Grant-in-Aid for Scientific Research (07672471) from the Ministry of Education, Science, and Culture and by Osaka City University Medical Research Foundation Fund for Medical Research.

Received April 12, 1996; first decision May 13, 1996; first decision October 7, 1996;
*    References
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