Calorie restriction increases telomerase activity

Authors: Naoki Makino Junichi Oyama Toyoki Maeda Masamichi Koyanagi Yoshihiro Higuchi Keiko Tsuchida

Publish Date: 2015/02/07

Volume: 403, Issue: 1-2, Pages: 1-11

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Abstract

The aims of this study were to investigate the impact of caloric restriction CR on cardiac telomere biology in an animal model of diabetes and to examine the signal transduction involved in cell senescence as well as cardiac function Male 8weekold Otsuka LongEvans Tokushima fatty OLETF diabetic rats were divided into two groups a group fed ad libitum OLETFAL and a group fed with CR OLETFCR 30  energy reduction LongEvans Tokushima Otsuka LETO nondiabetic rats were used as controls LETO rats were also divided into two groups a CR LETOCR group and a group fed AL LETOAL At 40 weeks of age the body weight was decreased by 97  and the insulin resistance was less in OLETFCR rats Telomerase activity in OLETFCR rats was significantly increased and telomerase reverse transcriptase was more highly expressed in those rats However the telomere length TL was not different between AL and CRtreated rats of each strain The protein expressions for FoxO1 and FoxO3 were increased in OLETFAL rats but the levels of phosphorylated pAkt were decreased compared to those in OLETFCR rats Autophagic LC3II signals revealed significant increases in OLETFCR rats Echocardiography showed that OLETFCR improved the left ventricular diastolic dysfunction without changes in the left ventricular dimension This study revealed that CR increases cardiac telomerase activity without TL attrition and significantly ameliorates diastolic dysfunction These findings suggest that cardiac telomerase activity may play an important role in the maintenance of normal cardiac functionIn a previous study using a rat model of type II diabetes mellitus DM rats model diabetic cardiomyopathy was characterized functionally by the presence of left ventricular LV diastolic dysfunction and histologically by interstitial fibrosis and collagen accumulation 1 Although coronary artery diseases are the main cause of heart failure and deteriorating function the high incidence of diabetic cardiomyopathy indicates that diabetes itself is an important factor in myocardial damage Conventional therapeutic practices such as strict control of blood glucose level and avoidance of traditional risk factors are often effective but not completely prevent cardiac complications 2 Calorie restriction is commonly recommended for prevention and amelioration of diabetes Caloric restriction CR has various beneficial effects on health including lifespan prolongation 3 One possible mechanism of the beneficial effects of CR is attenuation of mitochondrial dysfunction under various pathological conditions 4 Several studies conducted on laboratory rodents have shown that CR promotes longevity and ameliorates the ageassociated impairment of LV diastolic function arterial elasticity and heart rate variability 5 6In recent years the role of telomere length TL in the pathogenesis of cardiovascular disease and diabetes as attracted increasing research interest Telomeres the tandem repeats of the TTAGGG DNA sequence extending at the end of the eukaryotic chromosomes undergo attrition during every cell division and their length is the best indicator of the replication potential of somatic cells 7 8 9 The present work offers a proof of concept supporting the validity of the model that the difference between muscle TL and TL of proliferative tissues that is leukocytes or skin provides information over and above that of crosssectional analysis of agedependent TL shortening in proliferating tissuesWhile the mammalian heart has low but functionally significant levels of telomerase expression the cellular population responsible remains incompletely characterized 10 Emerging evidence also indicates that telomerase activity plays an important role in the development and function of the normal heart TERC −/− mice that lack telomerase activity develops cardiac abnormalities including dilated cardiomyopathy and reduced angiogenic potential 11 We previously observed that cardiac telomerase activity was associated with the insulin resistance in an experimental rat model of diabetes 12Aging is known to increase the prevalence of metabolic disorders such as diabetes 8 Cellular aging is considered to influence insulin resistance and this can lead to excessive calorie intake obesity and diabetes It is now apparent that cellular senescence can be induced by various stressors that are independent of cell replication such as chromatin damage related to oxidative stress Furthermore accumulating evidence also suggests a potential relationship between cellular senescence and the aging of organisms 13 Autophagy is also involved in various pathophysiological processes and shows increased activity in response to extracellular and intracellular stimulations such as CR Autophagy increases in response to acute and chronic myocardial ischemia heart failure and cardiomyopathic degeneration 14The effect of CR on telomerase in the heart has not previously been reported and here we tested the hypotheses that longterm CR can affect telomere biology as well as diastolic function in diabetes using a rat model of spontaneous type II diabetes Otsuka LongEvans Tokushima fatty OLETF rats OLETF rats develop hyperglycemic obesity with hyperinsulinemia and insulin resistance after the age of 25 weeks similar to patients with noninsulindependent DM 1 To gain a better understanding of the molecular mechanisms we also examined whether CR could modify the expression of senescencerelated molecules to ensure telomere integrity and thereby maintain tissue homeostasis and regulate lifespanExperiments using animal subjects or tissues from animals were conducted in accordance with the Guide for the Use and Care of Laboratory Animals NIH Pub No 85–23 Revised 1996 and were approved by the Institutional Animal Care and Use Committee of Kyushu University OLETF rats established genetic diabetic model of human type II 1 were used for all experiments in this study Male OLETF rats at 5 weeks of age were supplied by the Tokushima Research Institute Otsuka Pharmaceutical Tokushima Japan and upon arrival were randomly assigned to sedentary cage conditions with ad libitum feeding for 3 weeks At 8 weeks of age OLETF rats were divided into two groups one group was maintained for 32 weeks under cage conditions with ad libitum feeding OLETFAL and the other group was maintained under the same conditions but with food intake reduced to 70  of normal OLETFCR The daily amount of food intake was assessed in the rats with ad libitum feeding and was adjusted in the CR rats according to increasing body weight to maintain a food intake of 70  of ad libitum feeding Nonhyperphagic controlstrain LongEvans Tokushima Otsuka LETO rats were used as controls and were maintained under sedentary cage conditions The LETO rats were also divided into two groups a group with ad libitum feeding LETOAL and a CR group with food intake reduced to 70  of normal LETOCR All rats had free access to standard laboratory chow MF Oriental Yeast Tokyo Japan and tap water and were taken care of under the specifications outlined in the Guiding Principles for the Care and Use of Laboratory Animals All rats were caged individually in an environment in which temperature 23 ± 2 °C and humidity 55 ± 5  were controlled and an artificial light cycle was used During the study period both body weight and systolic blood pressure were measured by the tailcuff method BP98A Softran Tokyo Japan once a week For the analysis of blood samples blood was collected from the tail vein between 1000 AM and noon under nonfasting conditions during the study All serum samples were stored at −80 °C until analysis Plasma glucose levels were measured using the glucose oxidase method Plasma insulin levels and serum adiponectin levels were measured using an ELISA kit Linco Research St Charles MO As a marker of insulin resistance the homeostasis model assessment of insulin resistance HOMAIR which was determined based on both plasma glucose and serum insulin levels was analyzed At the end of the study the animals were killed by decapitation The liver and heart tissues were then excised All excised tissues were immediately frozen in liquid nitrogen and stored at −80 °C until used for analysisEach rat was lightly sedated with 3–4  isoflurane and then the chest was shaved and the rat was placed in the supine position After anesthesia with intraperitoneal sodium pentobarbital 50 mg/kg echocardiography was performed using an LOGIQ 400 PRO ultrasonography system GE Medical Systems Milwaukee WI equipped with a 10–12 MHz transducer as previously described 12 15 The transmitral flow velocity profile was determined by positioning a sample volume at the tip of the mitral valve on the apical four chamber view The Doppler beam was set within 15° of the incident angle to the flow direction identified on the color Doppler image Angle correction was performed by a semiautomated system installed in the machine The peak velocity E deceleration time DT of the early diastolic filling wave and early mitral annulus velocity E′ were measured 16 17 DT was obtained by extrapolating the initial slope of early diastolic filling wave deceleration to the baseline 1 E′ was measured at the septal portion of the mitral annulus in an apical four chamber view using a tissue Doppler technique with a Nyquist limit of 15 cm/s The transmitral inflow pattern was recorded on a strip chart at 100 mm/s sweep speed with simultaneous 3lead electrocardiography for offline analysis All measurements represented the mean of 5 consecutive cardiac cycles and heart rate was calculated on the basis of the strip chart of Doppler echocardiography

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