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. 2010 Winter;15(4):e128–e133.

Telomere biology in cardiovascular disease – role of insulin sensitivity in diabetic hearts

Naoki Makino 1,✉, Makoto Sasaki 1, Toyoki Maeda 1, Kosi Mimori 2
PMCID: PMC3016072  PMID: 21264070

Abstract

The present study examined telomere biology in the context of insulin sensitivity in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type 2 diabetic animal model. To improve insulin sensitivity, pioglitazone (PG; 10 mg/kg/day) was administered to OLETF rats between 20 weeks and 40 weeks of age, and the effects of the treatment were compared with those in untreated OLETF rats or control Long-Evans Tokushima Otsuka rats. The homeostasis model assessment of insulin resistance index significantly increased in OLETF rats, but decreased in PG-treated OLETF rats. Telomere lengths were not shortened in heart tissues of OLETF rats; however, telomerase activity was decreased in heart tissues of OLETF rats. Messenger RNA expression for both telomerase reverse transcriptase and telomere repeat binding factor 2 was downregulated in the hearts of OLETF rats. Protein expression of phosphorylated Akt, insulin-like growth factor and endothelial nitric oxide synthase were all reduced in OLETF rats. The changes observed in OLETF rats were inhibited by PG treatment. Myocardial fibrosis was less extensive and diastolic dysfunction improved in PG-treated OLETF rats versus untreated OLETF rats. These findings suggest that improving insulin sensitivity via the activation of peroxisome proliferator-activated receptor-gamma may exert regulatory effects on cardiac telomere biology, and may have desirable morphological and functional effects on the diabetic heart.

Keywords: Diabetes, Pioglitazone, PPAR-γ, Remodelling, Telomere

Telomeres are repetitive DNA sequences coated by capping proteins at the ends of linear chromosomes. In human cells, telomeres consist of hundreds to thousands of TTAGGG tandem repeats in the leading strand (1). A single-stranded 3′-hydroxyl overhang is generated by the catalytic addition of telomeric repeats to the 3′ end and by postreplicative processing of the lagging strand. Capping proteins, which coat the telomeric DNA sequence (2), serve as a molecular signal to prevent the cellular DNA repair machinery from mistaking telomeres for double-stranded DNA breaks. When they are too short, telomeres signal the arrest of cell proliferation, and trigger senescence and apoptosis. This process explains the interruption of proliferation in cultured human cells (3). Telomere length has been associated with cardiovascular complications, but the associations have varied across studies; exploratory epidemiological surveys often do not correct for multiple variables, and there is no accepted pathophysiological link (4,5). In one study (6), endothelial progenitor-cell telomeres were shorter in patients with coronary artery disease than in healthy persons, and intensive lipid-lowering therapy reduced oxidative DNA damage and also prevented further telomere attrition. In one of many epidemiological surveys (7), individuals with short leukocyte telomeres were found to be at risk for coronary artery disease, which appeared to be attenuated by statin therapy. In the Cardiovascular Health Study (8), shortened telomeres corresponded with a risk of myocardial infarction among younger patients that was three times as high as the risk among older patients. In the Heart and Soul Study (9), shorter-than-normal telomeres were a biomarker for the risk of death in patients with stable coronary artery disease. Telomere length was short in a study (10) involving British patients who experienced premature myocardial infarction. In the Framingham Heart Study (11), shortened telomere length correlated with carotid artery intimal thickening. The present study was conducted to examine whether improving insulin sensitivity using a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist (pioglitazone [PG]) would affect telomere length or telomerase activity in the liver, muscle, heart and fat tissues of type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Previous data (12) have demonstrated the presence of short telomeres limited to localization in peripheral leukocytes in type 2 diabetes. To gain a better understanding of the molecular mechanisms underlying telomere biology in the context of improved insulin resistance, we also analyzed gene products implicated in both growth arrest and cardiac function.

METHODS

Animal studies

Male OLETF rats were supplied by the Tokushima Research Institute (Otsuka Pharmaceutical Co, Japan) and were used as an animal model of type 2 diabetes (13). Age-matched male Long-Evans Tokushima Otsuka (LETO) rats served as the genetic control for OLETF rats. Rats at 20 weeks of age, ie, those at the prediabetic stage, were divided into three groups (six rats per group). Group 1 consisted of LETO control rats, group 2 consisted of OLETF rats with type 2 diabetes, and group 3 consisted of OLETF rats treated with 10 mg/kg of PG (Takeda Pharmaceutical Co, Japan) mixed with rat chow until 40 weeks of age (the age when OLETF rats usually enter the advanced stage of diabetes). At the end of the study, the animals were sacrificed by decapitation, and the liver, skeletal muscle, heart and white adipose tissues were excised. All excised tissues were immediately frozen in liquid nitrogen and stored at −80°C until they were used for analysis. Animal experiments conformed with the Guide for the Use and Care of Laboratory Animals (publication number 85-23; revised 1996) published by the United States National Institutes of Health. The experiments were conducted in accordance with the Kyushu University Guide for the Treatment of Laboratory Animals.

Genomic DNA extraction from rat tissue

Rat tissue samples were lysed by incubation at 55°C for 48 h in 200 μL of lysis buffer containing 10 mmol/L of Tris/HCl (pH 8.0), 0.1 mmol/L of EDTA (pH 8.0), 2% sodium dodecyl sulphate and 500 μg/mL of protease K (Roche Diagnostic, Japan). Genomic DNA extraction was performed using a DNeasy Tissue Kit (Qiagen KK, Japan) according to the manufacturer’s recommendations.

Dot blot analysis

The length of the telomeric DNA was estimated as the telomeric/centromeric DNA contents ratio, as previously reported (14). Telomeric DNA content can be standardized by calculating the relative telomeric DNA content with the centromeric DNA content (0.1 g). DNA samples were diluted and denatured. The telomere probe was visualized with CDP-Star (New England BioLabs Inc, USA), a highly sensitive, alkaline phosphatase-metabolizing chemiluminescence substrate.

Reverse transcription – polymerase chain reaction analysis

Total RNA was extracted using RNAzol B (Tel-Test Inc, USA). Reverse transcription-polymerase chain reaction (RT-PCR) was used to generate messenger RNA (mRNA) transcripts for PPAR-γ, telomerase reverse transcriptase (TERT), telomerase RNA (TERC) and terminal restriction fragment 2 (TRF2) and labelled using a DIG detection system (Roche Applied Science, Japan). Complementary DNA for each sample was produced by RT-PCR using primers derived from sequences specific to each rat strain. Primers for TERT (15) and TRF2 (16) were designed and used according to methods previously described in the literature. The values for TERT and TRF2 mRNA levels were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level in the same sample. For semiquantitative PCR, GAPDH was used as an internal control to evaluate total RNA input, as previously described.

Western blot and other analyses

Tissues were homogenized in five volumes of RIPA buffer, and the supernatants were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The membranes were blocked and subsequently incubated with antibodies against checkpoint-2 (Santa Cruz Biotechnology, USA), p-21 (Santa Cruz Biotechnology), phosphorylated Akt (phospho-Akt) (Cell Signaling, USA), insulin-like growth factor 1 (IGF-1) (Upstate, USA) or GAPDH (Imgenex Corp, USA). Detection was performed with secondary horseradish peroxidase-conjugated antibodies (Chemicon, USA) and the ECL Detection System (GE Healthcare, Japan), as previously described (14).

Telomerase activity

Telomerase activity was examined by means of a modified telomerase repeat amplification protocol method (14) with TeloChaser (Toyobo, Japan) according to the manufacturer’s instructions. A human cancer cell line overexpressing telomerase was used as the reference in each assay.

Echocardiography

The rats were anesthetized with ketamine (50 mg/kg, intraperitoneally) and xylazine (10 mg/kg, intraperitoneally); their body temperatures were maintained with a heat lamp. The animals chests were shaved, and the rats were placed in the left lateral decubitus position. Echocardiographic images were obtained by using the LOGIQ 400PRO system (GE Medical Systems, USA) equipped with a 10 MHz to 12 MHz transducer. The following parameters were used as indicators of diastolic ventricular function: peak transmitral flow velocity in early diastole (E), peak transmitral flow velocity in late diastole (A), E/A ratio and deceleration time of the E-wave. The left ventricular end-diastolic (LVEDD) and end-systolic (LVESD) dimensions were also measured on M mode echocardiograms. The fractional shortening was calculated from the LVEDD and LVESD echocardiogram data.

Histological and other examinations

To measure the fibrotic area, heart tissues (six per group) were fixed with 10% buffered formalin and subsequently embedded in paraffin and stained with Masson’s trichrome stain. Images of the left ventricular area of each slide were prepared using an Olympus BX41 system (original magnification ×20; Olympus Corporation, Japan) with a charge-coupled device camera. The quantification of fibrosis was performed by determining the myocardial collagen concentration, measured as the hydroxyproline concentration, as previously described (17).

Statistical analysis

The intergroup comparisons were performed using an independent samples t test and one-way ANOVA. Paired samples were compared using the paired t test. P<0.05 was considered to be statistically significant. Group data were expressed as mean ± SD. The statistical analyses were performed using SPSS version 10.0 software (Microsoft Corp, USA).

RESULTS

General characteristics of the experimental rats used in the present study are shown in Table 1. At 40 weeks of age, the body weight of OLETF rats significantly increased (P<0.05) compared with that of LETO rats. PG-treated OLETF rats showed a significantly greater increase (P<0.05) in body weight than LETO rats, but did not differ from untreated OLETF rats. The homeostasis model assessment of insulin resistance (HOMA-IR) index was determined based on both plasma glucose and serum insulin levels and was significantly more elevated in OLETF rats than in LETO rats; however, the HOMA-IR index was reduced by PG treatment. Plasma glucose levels were significantly increased in OLETF rats at 20 weeks of age compared with LETO rats, but plasma glucose levels of PG-treated OLETF rats were significantly reduced compared with untreated OLETF rats. The concentration of serum adiponectin was more elevated in PG-treated rats than in either untreated OLETF or LETO rats. Telomeric DNA length was assessed by dot blot analysis of tissues of 40-week-old rats (Figure 1). The length of the telomeric DNA was significantly shorter in the liver tissue of OLETF rats, but remained unaltered in the heart, skeletal muscle and fat tissues. After PG treatment, telomere length in the liver tissue was not significantly shortened, nor was it different in other tissues examined after PG treatment.

TABLE 1.

General characteristics

LETO, weeks
 OLETF, weeks
 OLETF + PG, weeks
20 40 20 40 20 40
Body weight, g 445±42 508±49 619±18 715±15 585±48 676±66*
Plasma glucose, mg/dL 131±5.0 137±4.6 173±9.8 189±9.6 168±10.4 149±10*†
Serum insulin, μU/mL 3.10±1.24 3.13±1.63 7.61±1.42 8.20±1.00 8.05±0.76 4.43±11*†
HOMA-IR 1.14±0.38 1.05±0.52 3.28±0.29 3.49±0.35 3.26±0.39 1.63±1.4*†
Adiponectin, mg/mL 2.07±0.14 1.91±0.12 1.76±0.15 1.82±0.14 1.90±0.12 5.74±0.32*†
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Data are presented as mean ± SD.

*

P<0.05 versus Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 40 weeks of age;

†

P<0.05 versus pioglitazone (PG)-treated OLETF rats at 20 weeks of age. HOMA-IR Homeostasis model assessment of insulin resistance; LETO Long-Evans Tokushima Otsuka

Figure 1).

Figure 1)

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The length of telomeric DNA in tissue from Long-Evans Tokushima Otsuka (LETO), Otsuka Long-Evans Tokushima Fatty (OLETF) and pioglitazone (PG)-treated OLETF rats. The length of telomeric DNA, assessed by dot blot analysis, is presented as the telomeric/centromeric (T/C) DNA content ratio. Data were obtained from liver, heart, skeletal (Sk) muscle and fat tissues of LETO, OLETF and PG-treated OLETF rats. Representative dot blot data are shown in the left panel. The relative T/C ratio is given in the right panel, which shows data from LETO (white column), OLETF (black column) and PG-treated OLETF (grey column) rats. Each group contained six animals. Data are presented as mean ± SD

Thus, telomere length differed in each organ of 40-week-old diabetic animals, and PG treatment inhibited telomere shortening in liver tissue specifically. Telomerase activity, quantified using a telomere repeat amplification protocol assay, was found to be significantly decreased in the liver, heart and fat tissues of OLETF rats, but was not decreased in the liver and heart tissues of PG-treated OLETF rats (Figure 2). However, telomerase activity in fat tissue was reduced in OLETF rats and did not appear to be sensitive to PG treatment. To evaluate telomerase activity in the heart of experimental rats, mRNA levels of the catalytic subunits TERT and TERC were assessed by RT-PCR (Figure 3A). The expression of TERT mRNA was attenuated in OLETF rats, but not in PG-treated OLETF rats. Similar results were obtained for telomere-associated protein TRF2. The expression of TERT and PPAR-γ mRNA did not change in the three experimental rat groups. Thus, mRNA expression for both TERT and TRF2 was not attenuated in PG-treated OLETF rats, which was also observed in LETO rats (Figure 3B). TRF1 mRNA expression levels did not differ among the experimental groups in the study (data not shown).

Figure 2).

Figure 2)

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Telomerase activity in liver, heart and fat tissues from Long-Evans Tokushima Otsuka (LE), Otsuka Long-Evans Tokushima Fatty (OL) and pioglitazone (PG)-treated OL (OL+PG) rats. Representative results are shown in panel A. The summarized results are shown in panel B. Each bar indicates the same pattern as in Figure 1. Each group contained six animals. Data presented as mean ± SD. *P<0.05 versus LETO rats; †P<0.05 versus untreated OLETF rats. MW Molecular weight; NC Negative control; PC Positive control

Figure 3).

Figure 3)

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Messenger RNA (mRNA) expression for peroxisome proliferator-activated receptor-gamma (PPAR-γ), telomere reverse transcriptase (TERT), telomere RNA component (TERC) and telomere repeat-binding factor 2 (TRF2) from the heart of Long-Evans Tokushima Otsuka (LE), Otsuka Long-Evans Tokushima Fatty (OL) and pioglitazone (PG)-treated OL (OL+PG) rats were determined by reverse transcription-polymerase chain reaction. Representative results are shown in panel A, and the summarized results are shown in panel B. Each bar indicates the same pattern as in Figure 1. Each group contained six animals. Data presented as mean ± SD. *P<0.05 versus LETO rat; †P<0.05 versus untreated OL rats

The expression of the cell signalling survival factors phospho-Akt, IGF-1 and endothelial nitric oxide synthase in the tissues of the experimental animals was investigated (Figure 4). Western blot analysis showed that expression of these proteins was significantly reduced in the hearts of OLETF rats, but not in hearts of PG-treated rats. To further examine the function of telomere-associated protein complexes in the context of improving insulin sensitivity, the DNA damage checkpoint protein kinases p21 and checkpoint-2, and p53 – a proapoptotic marker – were assessed by Western blot analysis. The expression of these factors was enhanced in the hearts of OLETF rats, and expression was significantly attenuated in PG-treated OLETF rats. Echocardiographic analyses of the three groups of rats at 40 weeks of age were also conducted (Figure 5). Deceleration time of the E-wave was significantly prolonged in untreated OLETF rats compared with LETO rats. This parameter was reduced in PG-treated rats and was not different from LETO rats. These results indicate that diastolic relaxation was impaired in OLETF rats, and PG treatment had protective effects in treated rats. LVEDD was significantly increased in 40-week-old OLETF rats; however, both LVESD and fractional shortening were not different among those experimental rats. A histological examination was also performed in the present study; representative photomicrographs of the heart are shown in Figure 6. More prominent cardiac fibrosis, as assessed by Masson’s trichrome staining, was observed in the hearts of OLETF rats than in either LETO rats or PG-treated OLETF rats. To quantify the extent of myocardial fibrosis in experimental rats, the collagen concentration was estimated by measurement of the hydroxyproline content in heart tissue. The collagen concentration was significantly higher in OLETF rat hearts than in LETO rat hearts (2.21±0.61 mg/mg dry tissue weight versus 4.03±0.78 mg/mg dry tissue weight, respectively; P<0.05). Moreover, the collagen concentration was significantly reduced in PG-treated OLETF rats (2.74±0.73 mg/mg wet weight) compared with OLETF rats.

Figure 4).

Figure 4)

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Western blot analysis of protein expression for p21, checkpoint kinase 2 (Chk2), p53, endothelial nitric oxide synthase (eNOS), phosphorylated-Akt (p-Akt) and insulin-like growth factor 1 (IGF-1) in the hearts of Long-Evans Tokushima Otsuka (LE), Otsuka Long-Evans Tokushima Fatty (OL) and pioglitazone (PG)-treated OL (OL+PG) rats. Representative results are shown in panel A, and the summarized results are shown in panel B. Each bar indicates the same pattern as in Figure 1. Each group contained six animals. Data presented as mean ± SD. *P<0.05 versus LE rats, †P<0.05 versus untreated OL rats. GAPDH Glyceraldehyde 3-phosphate dehydrogenase; mRNA Messenger RNA

Figure 5).

Figure 5)

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Echocardiographic findings in Long-Evans Tokushima Otsuka (LE), Otsuka Long-Evans Tokushima Fatty (OL) and pioglitazone (PG)-treated OL (OL+PG) rats at 40 weeks of age. Percentage of fractional shortening (FS), left ventricular end-diastolic (LVDd) and deceleration time of the E-wave (DcT) were measured on M mode echocardiograms. Each bar indicates the same pattern as in Figure 1. *P<0.05 versus LE rats; †P<0.05 versus untreated OL rats

Figure 6).

Figure 6)

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Cardiac fibrosis in Long-Evans Tokushima Otsuka (LETO), Otsuka Long-Evans Tokushima Fatty (OLETF) and pioglitazone (PG)-treated OLETF (OLETF + PG) rats at 40 weeks of age. Each panel shows a representative transverse axis image of fibrosis visualized by Masson’s trichrome staining. Original magnification × 100. The bar graph shows the collagen concentration (mg/mg wet weight) in the left ventricle of five animals. Data are presented as mean ± SD. *P<0.05 versus LETO rats; †P<0.05 versus untreated OLETF rats

DISCUSSION

We examined the effects of a PPAR-γ agonist on telomere biology in OLETF rats, an animal model of diabetes. Adult animals at 40 weeks of age showed high HOMA-IR, a marker of insulin resistance. Reduced telomere length was detected in the liver, but not in the heart, of OLETF rats. Telomerase activity was inhibited, and the mRNA expression of the telomere-associated proteins TERT and TRF2 was also downregulated in the heart tissue of OLETF rats. These effects were inhibited by treatment with a PPAR-γ agonist, PG (ie, expression levels were reversed to those observed in LETO rats). These findings support the hypothesis that PPAR-γ activation confers favourable effects on cardiac telomerase in rats with type 2 diabetes.

It was recently reported that hypertension, insulin resistance and oxidative stress are associated with relatively short telomere length in human leukocytes (18). Moreover, telomere length was found to be inversely related to age; age-adjusted telomere length was observed to be shorter in individuals with cardiovascular disease than in healthy individuals (19). The present study addressed the heart, liver, skeletal muscle and fatty tissues of male OLETF rats; however, leukocytes were not considered. Comparison with LETO rats revealed telomere shortening only in the liver, but not in tissues from other organs of OLETF rats. In light of discrepant results regarding telomere length, it appears that the rate of telomere shortening is not consistent across organs in aged animals because telomere length in somatic cells reflects replicative history and is predictive of remaining proliferative potential. Thus, the rate of telomere shortening in each organ may be age dependent. Telomeres consist of tandem repeats at the ends of chromosomes, and are maintained by a catalytic subunit of TERT, and bound by specific TRFs including TRF1 and TRF2 (19,20). In the present study, telomerase activity was decreased in the liver, heart and fat tissues of OLETF rats, as shown in Figure 2. Compared with that of LETO rats, the expression of TRF2 and TERT mRNAs was downregulated in OLETF rats at 40 weeks of age (Figure 3) – an advanced diabetic stage – but these observations were not the case with TRF1. However, PG treatment led to the recovery of mRNA expression in the heart tissue to levels similar to those observed in LETO rats. TRF2 and TERT are both able to bind to the TTAGGG repeats of telomeres and both contribute to the formation of chromosome-protecting T loops (21). Thus, telomere-associated proteins are believed to be important for regulating cardiac muscle cell growth and survival (22).

In the present study, we observed inhibition of expression of survival factors promoting cell growth and survival, ie, factors such as IGF-1 and phospho-Akt, which may delay cellular aging in diabetes. It has been firmly established that IGF-1 protects cardiomyocytes from apoptotic cell death (23). IGF-1 reduces myocyte senescence and death, and telomerase activity is enhanced in younger myocytes, in which this enzyme is protective against telomere shortening, oxidative cellular injury, growth arrest and cell death. The elevated expression of IGF-1 proteins implies an active process of DNA repair and telomere integrity in cardiomyocytes. Another survival factor, phospho-Akt, possesses the ability to activate telomerase via phosphorylation of the enzyme, and can thereby promote myocyte growth and delay the onset of myocyte aging. Thus, both signals may affect the preservation of TERT and TRF2 in myocytes. Furthermore, the expression levels of proteins for both signals were decreased in OLETF rats, as was endothelial nitric oxide synthase, and levels were enhanced to those observed in LETO rats. The present data identified IGF-1, phospho-Akt and endothelial nitric oxide synthase as important mediators of pathology in the diabetic heart. Thus, in diabetes at the adult stage, these cell survival factors are all downregulated, but were inhibited via activation of a PPAR-γ agonist. The current results demonstrate that PG improves left ventricular diastolic function in OLETF rats with type 2 diabetes (24). Although the precise mechanism by which improving insulin resistance remediates the left ventricular diastolic function associated with diabetes remains unknown, the amelioration of insulin resistance may be among the mechanisms responsible for diastolic functional recovery in the diabetic heart. In addition, myocardial fibrosis is a factor known to contribute to the functional changes induced by diabetes.

CONCLUSION

PPAR-γ treatment was found to be effective in elevating telomerase activity and the expression of telomere-associated proteins as well as myocardial contractile function in OLETF rats. Ultimately, the results of the present study indicated that PPAR-γ agonists may play potential roles as therapeutic agents in postponing the aging process in patients with type 2 diabetes.

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