The Relationship Between Dietary Macronutrients and Hepatic Telomere Length in Aging Mice

The Relationship Between Dietary Macronutrients and Hepatic Telomere Length in Aging Mice Abstract Macronutrients and dietary energy influence aging, age-related health, and life span. Reduction in telomere length has been proposed as one mechanism for aging. Therefore, this study investigated the effects of varying ratios of dietary macronutrients and energy on telomere length in older adult mice. C57Bl/6 mice were fed ad libitum their entire life on one of 25 diets varying in protein, carbohydrates, fat, and energy. Average telomere length ratio (ATLR) was measured by polymerase chain reaction in livers of a subset of 161 mice aged 15 months. There was a significant positive relationship between ATLR and carbohydrate intake and a negative relationship with protein intake, but no relationships with fat or energy intake. Analysis using the Geometric Framework and Generalized Additive Models confirmed that carbohydrate intake was positively associated with ATLR, while the longest ATLR was achieved by mice restricted to low protein, high carbohydrate diets. ATLR distribution across the diets was parallel to median life-span results previously published. ATLR was associated with blood levels of some amino acids (asparagine, glutamate, taurine) but not with blood levels of fatty acids, hepatic mitochondrial function, or nutrient sensing pathways. In conclusion, mice on low protein, high carbohydrate diets have the longest hepatic telomeres and longest life span. Diet, Nutrition, Telomeres, Geometric Framework, Macronutrients Aging can be delayed by a variety of nutritional interventions, of which caloric restriction has been most extensively studied (1). Recent studies utilizing the principles of nutritional geometry have found that dietary macronutrients also influence life span, with most studies in insects and mice reporting that low protein, high carbohydrate diets maximize life span in ad libitum-fed animals (2,3). In a recent study, mice maintained on diets with a protein to carbohydrate ratio of nearly 1:10 had the longest life span and best latelife health despite increased fat mass (3). These diets were associated with optimization of various nutrient-sensing pathways that influence aging including mTOR, insulin (3), FGF21, and IGF-1 (4). A large number of biological processes underlie aging and these have been termed the “hallmarks of aging” (5). Among these processes is telomere attrition. Telomeres form the ends of eukaryotic chromosomes and comprise repetitive stretches of DNA (TTAGGG) bound to specific proteins. Telomeres shorten with each mitotic cell division, eventually leading to replicative arrest and cellular senescence (6). Although the results are variable, a number of epidemiological studies have suggested that leukocyte telomere length (LTL) decreases with age in humans (7–9). Thus, telomere length has been proposed to be a biomarker of aging, with short telomeres contributing to aging by causing cellular senescence (6,7). Here, we investigated whether dietary macronutrients influence telomere length in aging mice. The relationship between telomere length and various markers of nutrition (nutrient sensing pathways, circulating amino acids and fatty acids) and mitochondrial function were also explored as potential mechanistic links between nutrition, telomeres, and aging. Methods The life span, metabolomics, and signaling pathway data from this mouse study have been published previously (3,4). Briefly, 3-week-old male and female C57Bl6/J mice (n = 858) were provided ad libitum access to one of 25 experimental diets varying systematically in protein (5%–60%), fat (16%–75%), carbohydrate (16%–75%), and energy (8, 13, or 17 kJ/g of food). At 15 months of age, a subset of mice was euthanized and tissues collected, while the remaining mice were maintained on their diets for life-span determination. Blood levels of insulin, FGF21, and IGF-1 were measured by ELISA, hepatic mitochondrial function using Seahorse XF Extracellular Flux Analyzer, hepatic mTOR and p-mTOR by western blotting and blood levels of amino acids and fatty acids by the Australian Proteomic Analysis Facility as described previously (3,4). The study was approved by the Sydney Local Health District Animal Welfare Committee (protocol no. 2009/003). Average telomere length was measured from liver DNA using a real-time quantitative polymerase chain reaction method (10). Frozen liver tissue samples were sectioned into 10 mg blocks and homogenized using Qiagen TissueLyser LT (Qiagen, Victoria Australia). DNA, RNA, and protein were extracted according to the protocol listed in the Qiagen AllPrep DNA/RNA/Protein Mini Handbook. DNA Forward and reverse telomere primers were 5′ CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3′ and 5′ GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 3′. Forward and reverse primers for the control 36B4 gene were 5′ ACT GGT CTA GGA CCC GAG AAG 3′ and 5′ TCA ATG GTG CCT CTG GAG ATT 3′ (11). A pooled sample of mouse DNA from all samples was serially diluted from 1 to 100 ng per well to serve as references for standard curves. Standard curves for 36B4 and telomeric repeats were used for absolute quantitation of samples using LightCycler software. Telomere concentrations were normalized to the 36B4 control gene concentration. Data obtained in this manner from replicates are considered to be the average telomere length ratio (ATLR) (11). Dietary macronutrients were expressed as kJ/mouse/day. Data were analyzed using Pearson’s correlation coefficient (Sigmaplot v11 Systat Software Inc, Germany). The Geometric Framework for Nutrition (GFN) was used to visualize the relationship between macronutrients and ATLR using thin-plate splines, with statistical significance calculated using Generalized Additive Models (GAMs) in R (12) as described previously (3,4). The GFN is a multidimensional approach for interpreting the relationships between nutrients, energy, the interactions between nutrients, and phenotype (13–15). p values less than .05 were considered statistically significant and a Bonferroni correction was applied for multiple comparisons. Results Telomere length was able to be determined in DNA extracted from 161 livers collected from 183 mice. The average ATLR was 6.8 ± 18.2, the median 2.0, and the majority of the values were between 1 and 4 (Figure 1A). There was no difference between females (n = 85, 7.1 ± 16.6) and males (n = 76, 6.4 ± 14.5) so they were pooled. The telomere length in kbp was estimated from the ATLR using an empiric formula derived in C57Bl/6 mice published elsewhere (11). Using this estimation, the telomere length across all diets averaged 16.6 ± 23.3 kbp and the distribution of telomere length was skewed to the right (Figure 1A and B). Figure 1. View largeDownload slide The distribution of telomere lengths in DNA extracted from mouse livers, determined by ATLR (A) and converted to kbp (B) according to the empiric equation of Callicott and Womack (1). The y-axis refers to the number of mice. Figure 1. View largeDownload slide The distribution of telomere lengths in DNA extracted from mouse livers, determined by ATLR (A) and converted to kbp (B) according to the empiric equation of Callicott and Womack (1). The y-axis refers to the number of mice. There was a significant positive correlation between carbohydrate intake and ATLR (r = .18, p = .02) and a significant negative correlation with protein intake (r = −.16, p = .04). There were no correlations between ATLR and fat intake or total energy intake (Supplementary Figure 1A–D). Analysis of the full, 3-macronutrient response surface using GFN and GAMS revealed that carbohydrate intake was the main driver of ATLR (p = .04), with the longest telomeres occurring in mice on the low protein, high carbohydrate diets. The pattern of distribution of ATLR across the dietary surfaces matched the changes we previously reported for life span, such that the longest telomeres and life span were seen in mice maintained on low protein high, carbohydrate ad libitum-fed diets (Figure 2A and B, Supplementary Table 1). We could not directly compare ATLR and life span in the same animals because ATLR was measured in animals sacrificed at 15 months of age. Figure 2. View largeDownload slide Representation of the relationship between macronutrients and ATLR (A) and median life span (B) using the Geometric Framework. Three 2D slices are given to show all three nutrient dimensions (protein, carbohydrate, fat). For each 2D slice, the third factor is at its median (shown below the x-axis in parentheses). In all surfaces, red indicates the highest value, while blue indicates the lowest value with the colors standardized across the three slices. The diet with the longest telomere length and life span is demonstrated with the red line, and this was a low protein, high carbohydrate diet in both cases. There is a very similar response of both average telomere length ratio and median life span to dietary macronutrients. The median life span data are reproduced with permission from Cell Metabolism as published previously by Solon-Biet et al. (3). Figure 2. View largeDownload slide Representation of the relationship between macronutrients and ATLR (A) and median life span (B) using the Geometric Framework. Three 2D slices are given to show all three nutrient dimensions (protein, carbohydrate, fat). For each 2D slice, the third factor is at its median (shown below the x-axis in parentheses). In all surfaces, red indicates the highest value, while blue indicates the lowest value with the colors standardized across the three slices. The diet with the longest telomere length and life span is demonstrated with the red line, and this was a low protein, high carbohydrate diet in both cases. There is a very similar response of both average telomere length ratio and median life span to dietary macronutrients. The median life span data are reproduced with permission from Cell Metabolism as published previously by Solon-Biet et al. (3). The correlations between ATLR, circulating amino acids (n = 120) and fatty acids (n = 128) were assessed. There were significant positive correlations between ATLR and asparagine (p = .01), glutamate (p = .0006), and taurine (p = .008) (Figure 3A and D), but not with other amino acids or fatty acids. Only glutamate remained significant after Bonferroni correction for analysis of 26 amino acids (Supplementary Table 2). There was a weak association with one fatty acid C21.0 (p = .04). Figure 3. View largeDownload slide The relationship between circulating amino acids and average telomere length ratio. There were significant correlations with asparagine (A, r = .23, p = .01), glutamate (B, r = .31, p = .0006), taurine (C, r = .24, p = .008), and a weak correlation with total amino acids (D, r = .18, p = .05) but not with the other amino acids. Figure 3. View largeDownload slide The relationship between circulating amino acids and average telomere length ratio. There were significant correlations with asparagine (A, r = .23, p = .01), glutamate (B, r = .31, p = .0006), taurine (C, r = .24, p = .008), and a weak correlation with total amino acids (D, r = .18, p = .05) but not with the other amino acids. There were no statistically significant correlations between ATLR and hepatic pmTOR/mTOR, measures of hepatic mitochondrial function determined by Seahorse, or circulating levels of FGF21, insulin or IGF-1 (Supplementary Table 2). Discussion Many observational studies in human populations have found an association between various diet constituents and LTL. Consumption of antioxidant rich, plant-derived foods, and the Mediterranean diet are linked with longer LTL, while shorter LTL have been associated with dietary fats, refined cereals, meat products, and sugar-sweetened beverages (reviewed in refs. (7,16,17)). In a prospective study of young humans, change in telomere length over time was inversely associated with total energy intake as well as that of each macronutrient (18). A number of environmental and lifestyle factors including diet appear to influence telomere length, which may provide a mechanism for their effects on aging and age-related health (7,16). There have been fewer studies of nutrition and telomeres in mice and rats. Telomeres are longer and shorten more rapidly with ageing in rodents than in humans (19,20). Caloric restriction in mice was associated with longer LTL at 15 months (21) while 40% protein restriction maintained telomere length in livers of rats aged 16 months, compared with a decrease in telomere length in those rats on standard chow (20). In our study, in which mice had ad libitum access to food, telomere length assessed by ATLR was positively correlated with carbohydrate intake, and negatively correlated with protein intake, while fat and total energy intake had no effect. Analysis using the Geometric Framework showed that ATLR positively correlated with carbohydrate intake, and highest ATLR was achieved in mice maintained on low protein, high carbohydrate, low fat diets that were ad libitum-fed over a lifetime. The surface generated by Geometric Framework analysis reveals that ATLR doubled from approximately 6 in mice on high protein, low carbohydrate diets to about 13 in those on low protein high, carbohydrate diets. These values are approximately equivalent to 16 and 27 kbp (using an empiric conversion equation (11)), which are similar to values of 19 and 23 kbp seen in the livers of 16-month-old rats maintained on standard chow or protein restriction respectively (20). In the mouse caloric restriction study (21), liver telomere length was not reported, however, caloric restriction was associated with longer telomeres in kidney (94 kbp vs 62 kbp), lung (55 vs 21 kbp), and muscle (14 vs 11 kbp) at 15 months of age. Together, the data in humans and rodents are consistent with a robust effect of nutrition on telomere length in older age, thereby providing a plausible mechanistic link for the effects of nutrition on ageing and age-related health. Telomere length provides a biomarker and a mechanism for aging. The ability of telomere length to predict aging and life span has been reported both in aged mice (19,22,23) and humans (8,9). This relationship has mostly been studied using LTL (6). However, one study in humans found that telomere length in the liver was 12.9 kbp in newborns compared to 8.3 kbp in a centenarian (24), while there is a reduction in the percentage of longer telomeres in livers of rats by 15 months of age (22). In our study, we were not able to directly compare telomere length and life span in the same mice, because the telomeres were evaluated in the livers of mice sacrificed at 15 months of age. However, we were able to compare the responses of both ATLR and median life span to macronutrients in mice from the same experimental cohort. As shown in Figure 2, the surfaces demonstrating the relationship between macronutrients and median life span versus ATLR are almost identical. Mice maintained on low protein, high carbohydrate diets had the longest median life span and the longest ATLR. Our results provide indirect evidence that is consistent with the concept that long telomere length is associated with longer life span. In addition, the rankings by diet of the median life span were compared to the telomere length, and there was a significant positive relationship, such that diets associated with longest median life span were also associated with longest telomeres (Supplementary Figure 2). We then explored whether the association between telomeres, nutrition, and life span is mediated by several mechanisms that are thought to link nutrition with aging (25). There was no association with ATLR and circulating levels of insulin, FGF21 or IGF1, hepatic mTOR activation, nor hepatic mitochondrial activity, although this may reflect the power of the study. However, we did find an association between ATLR and blood levels of amino acids including asparagine, glutamate, and taurine. Previously it has been shown that the chronic circulating levels of most amino acids (except branched chain amino acids) are inversely correlated with protein intake (3). Therefore, the positive association between some amino acids and ATLR in this study may simply reflect the effects of a low protein diet, although only glutamate remained significant after correction for multiple comparisons. On the other hand, taurine is an antioxidant which could influence telomere shortening (26). There are few reports linking amino acids with telomere length. One report in humans studied the relationship between LTL and several circulating amino acids (alanine, glycine, histidine, phenylalanine, leucine, isoleucine, valine, and tyrosine) and found an inverse relationship with phenylalanine (27). In conclusion, mice maintained on low protein, high carbohydrate diets had hepatic longer telomeres which correlated with a longer median life span. Nutrition has a powerful impact on aging and age-related health, which might be mediated in part by its effects on telomere length. Supplementary Material Supplementary data is available at The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online. Funding Funding was from the Australian National Health and Medical Research Council (Project grant 571328, post-doctoral fellowship to S.S.-B.), the Ageing and Alzheimers Institute and the Sydney Medical School Foundation (post-graduate scholarship to R.G.). D.W. is partly supported by the American Australian Association Education Fund. Conflict of Interest None reported. References 1. Mercken EM, Carboneau BA, Krzysik-Walker SM, de Cabo R. Of mice and men: the benefits of caloric restriction, exercise, and mimetics. Ageing Res Rev . 2012; 11: 390– 398. doi:10.1016/j.arr.2011.11.005 Google Scholar CrossRef Search ADS PubMed  2. Le Couteur DG, Solon-Biet S, Cogger VCet al.   The impact of low-protein high-carbohydrate diets on aging and lifespan. Cell Mol Life Sci . 2016; 73: 1237– 1252. doi:10.1007/s00018-015-2120-y Google Scholar CrossRef Search ADS PubMed  3. Solon-Biet SM, McMahon AC, Ballard JWet al.   The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. Cell Metab . 2014; 19: 418– 430. doi:10.1016/j.cmet.2014.02.009 Google Scholar CrossRef Search ADS PubMed  4. Solon-Biet SM, Cogger VC, Pulpitel Tet al.   Defining the nutritional and metabolic context of FGF21 using the Geometric Framework. Cell Metab . 2016; 24: 555– 565. doi:10.1016/j.cmet.2016.09.001 Google Scholar CrossRef Search ADS PubMed  5. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell . 2013; 153: 1194– 1217. doi:10.1016/j.cell.2013.05.039 Google Scholar CrossRef Search ADS PubMed  6. Blasco MA. Telomeres and human disease: ageing, cancer and beyond. 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The rate of increase of short telomeres predicts longevity in mammals. Cell Rep . 2012; 2: 732– 737. doi:10.1016/j.celrep.2012.08.023 Google Scholar CrossRef Search ADS PubMed  20. Tanrikulu-Kucuk S, Ademoglu E. Dietary restriction of amino acids other than methionine prevents oxidative damage during aging: involvement of telomerase activity and telomere length. Life Sci . 2012; 90: 924– 928. doi:10.1016/j.lfs.2012.04.024 Google Scholar CrossRef Search ADS PubMed  21. Vera E, Bernardes de Jesus B, Foronda M, Flores JM, Blasco MA. Telomerase reverse transcriptase synergizes with calorie restriction to increase health span and extend mouse longevity. PLoS One . 2013; 8: e53760. doi:10.1371/journal.pone.0053760 Google Scholar CrossRef Search ADS PubMed  22. Cherif H, Tarry JL, Ozanne SE, Hales CN. Ageing and telomeres: a study into organ- and gender-specific telomere shortening. Nucleic Acids Res . 2003; 31: 1576– 1583. Google Scholar CrossRef Search ADS PubMed  23. Ludlow AT, Witkowski S, Marshall MRet al.   Chronic exercise modifies age-related telomere dynamics in a tissue-specific fashion. J Gerontol A Biol Sci Med Sci . 2012; 67: 911– 926. doi:10.1093/gerona/gls002 Google Scholar CrossRef Search ADS PubMed  24. Takubo K, Nakamura K, Izumiyama Net al.   Telomere shortening with aging in human liver. J Gerontol A Biol Sci Med Sci . 2000; 55: B533– 536. Google Scholar CrossRef Search ADS PubMed  25. Solon-Biet SM, Mitchell SJ, de Cabo R, Raubenheimer D, Le Couteur DG, Simpson SJ. Macronutrients and caloric intake in health and longevity. J Endocrinol . 2015; 226: R17– 28. doi:10.1530/JOE-15-0173 Google Scholar PubMed  26. Ozsarlak-Sozer G, Kerry Z, Gokce G, Oran I, Topcu Z. Oxidative stress in relation to telomere length maintenance in vascular smooth muscle cells following balloon angioplasty. J Physiol Biochem . 2011; 67: 35– 42. doi:10.1007/s13105-010-0046-2 Google Scholar CrossRef Search ADS PubMed  27. Eriksson JG, Guzzardi MA, Iozzo P, Kajantie E, Kautiainen H, Salonen MK. Higher serum phenylalanine concentration is associated with more rapid telomere shortening in men. Am J Clin Nutr . 2017; 105: 144– 150. doi:10.3945/ajcn.116.130468 Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences Oxford University Press

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Abstract

Abstract Macronutrients and dietary energy influence aging, age-related health, and life span. Reduction in telomere length has been proposed as one mechanism for aging. Therefore, this study investigated the effects of varying ratios of dietary macronutrients and energy on telomere length in older adult mice. C57Bl/6 mice were fed ad libitum their entire life on one of 25 diets varying in protein, carbohydrates, fat, and energy. Average telomere length ratio (ATLR) was measured by polymerase chain reaction in livers of a subset of 161 mice aged 15 months. There was a significant positive relationship between ATLR and carbohydrate intake and a negative relationship with protein intake, but no relationships with fat or energy intake. Analysis using the Geometric Framework and Generalized Additive Models confirmed that carbohydrate intake was positively associated with ATLR, while the longest ATLR was achieved by mice restricted to low protein, high carbohydrate diets. ATLR distribution across the diets was parallel to median life-span results previously published. ATLR was associated with blood levels of some amino acids (asparagine, glutamate, taurine) but not with blood levels of fatty acids, hepatic mitochondrial function, or nutrient sensing pathways. In conclusion, mice on low protein, high carbohydrate diets have the longest hepatic telomeres and longest life span. Diet, Nutrition, Telomeres, Geometric Framework, Macronutrients Aging can be delayed by a variety of nutritional interventions, of which caloric restriction has been most extensively studied (1). Recent studies utilizing the principles of nutritional geometry have found that dietary macronutrients also influence life span, with most studies in insects and mice reporting that low protein, high carbohydrate diets maximize life span in ad libitum-fed animals (2,3). In a recent study, mice maintained on diets with a protein to carbohydrate ratio of nearly 1:10 had the longest life span and best latelife health despite increased fat mass (3). These diets were associated with optimization of various nutrient-sensing pathways that influence aging including mTOR, insulin (3), FGF21, and IGF-1 (4). A large number of biological processes underlie aging and these have been termed the “hallmarks of aging” (5). Among these processes is telomere attrition. Telomeres form the ends of eukaryotic chromosomes and comprise repetitive stretches of DNA (TTAGGG) bound to specific proteins. Telomeres shorten with each mitotic cell division, eventually leading to replicative arrest and cellular senescence (6). Although the results are variable, a number of epidemiological studies have suggested that leukocyte telomere length (LTL) decreases with age in humans (7–9). Thus, telomere length has been proposed to be a biomarker of aging, with short telomeres contributing to aging by causing cellular senescence (6,7). Here, we investigated whether dietary macronutrients influence telomere length in aging mice. The relationship between telomere length and various markers of nutrition (nutrient sensing pathways, circulating amino acids and fatty acids) and mitochondrial function were also explored as potential mechanistic links between nutrition, telomeres, and aging. Methods The life span, metabolomics, and signaling pathway data from this mouse study have been published previously (3,4). Briefly, 3-week-old male and female C57Bl6/J mice (n = 858) were provided ad libitum access to one of 25 experimental diets varying systematically in protein (5%–60%), fat (16%–75%), carbohydrate (16%–75%), and energy (8, 13, or 17 kJ/g of food). At 15 months of age, a subset of mice was euthanized and tissues collected, while the remaining mice were maintained on their diets for life-span determination. Blood levels of insulin, FGF21, and IGF-1 were measured by ELISA, hepatic mitochondrial function using Seahorse XF Extracellular Flux Analyzer, hepatic mTOR and p-mTOR by western blotting and blood levels of amino acids and fatty acids by the Australian Proteomic Analysis Facility as described previously (3,4). The study was approved by the Sydney Local Health District Animal Welfare Committee (protocol no. 2009/003). Average telomere length was measured from liver DNA using a real-time quantitative polymerase chain reaction method (10). Frozen liver tissue samples were sectioned into 10 mg blocks and homogenized using Qiagen TissueLyser LT (Qiagen, Victoria Australia). DNA, RNA, and protein were extracted according to the protocol listed in the Qiagen AllPrep DNA/RNA/Protein Mini Handbook. DNA Forward and reverse telomere primers were 5′ CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3′ and 5′ GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 3′. Forward and reverse primers for the control 36B4 gene were 5′ ACT GGT CTA GGA CCC GAG AAG 3′ and 5′ TCA ATG GTG CCT CTG GAG ATT 3′ (11). A pooled sample of mouse DNA from all samples was serially diluted from 1 to 100 ng per well to serve as references for standard curves. Standard curves for 36B4 and telomeric repeats were used for absolute quantitation of samples using LightCycler software. Telomere concentrations were normalized to the 36B4 control gene concentration. Data obtained in this manner from replicates are considered to be the average telomere length ratio (ATLR) (11). Dietary macronutrients were expressed as kJ/mouse/day. Data were analyzed using Pearson’s correlation coefficient (Sigmaplot v11 Systat Software Inc, Germany). The Geometric Framework for Nutrition (GFN) was used to visualize the relationship between macronutrients and ATLR using thin-plate splines, with statistical significance calculated using Generalized Additive Models (GAMs) in R (12) as described previously (3,4). The GFN is a multidimensional approach for interpreting the relationships between nutrients, energy, the interactions between nutrients, and phenotype (13–15). p values less than .05 were considered statistically significant and a Bonferroni correction was applied for multiple comparisons. Results Telomere length was able to be determined in DNA extracted from 161 livers collected from 183 mice. The average ATLR was 6.8 ± 18.2, the median 2.0, and the majority of the values were between 1 and 4 (Figure 1A). There was no difference between females (n = 85, 7.1 ± 16.6) and males (n = 76, 6.4 ± 14.5) so they were pooled. The telomere length in kbp was estimated from the ATLR using an empiric formula derived in C57Bl/6 mice published elsewhere (11). Using this estimation, the telomere length across all diets averaged 16.6 ± 23.3 kbp and the distribution of telomere length was skewed to the right (Figure 1A and B). Figure 1. View largeDownload slide The distribution of telomere lengths in DNA extracted from mouse livers, determined by ATLR (A) and converted to kbp (B) according to the empiric equation of Callicott and Womack (1). The y-axis refers to the number of mice. Figure 1. View largeDownload slide The distribution of telomere lengths in DNA extracted from mouse livers, determined by ATLR (A) and converted to kbp (B) according to the empiric equation of Callicott and Womack (1). The y-axis refers to the number of mice. There was a significant positive correlation between carbohydrate intake and ATLR (r = .18, p = .02) and a significant negative correlation with protein intake (r = −.16, p = .04). There were no correlations between ATLR and fat intake or total energy intake (Supplementary Figure 1A–D). Analysis of the full, 3-macronutrient response surface using GFN and GAMS revealed that carbohydrate intake was the main driver of ATLR (p = .04), with the longest telomeres occurring in mice on the low protein, high carbohydrate diets. The pattern of distribution of ATLR across the dietary surfaces matched the changes we previously reported for life span, such that the longest telomeres and life span were seen in mice maintained on low protein high, carbohydrate ad libitum-fed diets (Figure 2A and B, Supplementary Table 1). We could not directly compare ATLR and life span in the same animals because ATLR was measured in animals sacrificed at 15 months of age. Figure 2. View largeDownload slide Representation of the relationship between macronutrients and ATLR (A) and median life span (B) using the Geometric Framework. Three 2D slices are given to show all three nutrient dimensions (protein, carbohydrate, fat). For each 2D slice, the third factor is at its median (shown below the x-axis in parentheses). In all surfaces, red indicates the highest value, while blue indicates the lowest value with the colors standardized across the three slices. The diet with the longest telomere length and life span is demonstrated with the red line, and this was a low protein, high carbohydrate diet in both cases. There is a very similar response of both average telomere length ratio and median life span to dietary macronutrients. The median life span data are reproduced with permission from Cell Metabolism as published previously by Solon-Biet et al. (3). Figure 2. View largeDownload slide Representation of the relationship between macronutrients and ATLR (A) and median life span (B) using the Geometric Framework. Three 2D slices are given to show all three nutrient dimensions (protein, carbohydrate, fat). For each 2D slice, the third factor is at its median (shown below the x-axis in parentheses). In all surfaces, red indicates the highest value, while blue indicates the lowest value with the colors standardized across the three slices. The diet with the longest telomere length and life span is demonstrated with the red line, and this was a low protein, high carbohydrate diet in both cases. There is a very similar response of both average telomere length ratio and median life span to dietary macronutrients. The median life span data are reproduced with permission from Cell Metabolism as published previously by Solon-Biet et al. (3). The correlations between ATLR, circulating amino acids (n = 120) and fatty acids (n = 128) were assessed. There were significant positive correlations between ATLR and asparagine (p = .01), glutamate (p = .0006), and taurine (p = .008) (Figure 3A and D), but not with other amino acids or fatty acids. Only glutamate remained significant after Bonferroni correction for analysis of 26 amino acids (Supplementary Table 2). There was a weak association with one fatty acid C21.0 (p = .04). Figure 3. View largeDownload slide The relationship between circulating amino acids and average telomere length ratio. There were significant correlations with asparagine (A, r = .23, p = .01), glutamate (B, r = .31, p = .0006), taurine (C, r = .24, p = .008), and a weak correlation with total amino acids (D, r = .18, p = .05) but not with the other amino acids. Figure 3. View largeDownload slide The relationship between circulating amino acids and average telomere length ratio. There were significant correlations with asparagine (A, r = .23, p = .01), glutamate (B, r = .31, p = .0006), taurine (C, r = .24, p = .008), and a weak correlation with total amino acids (D, r = .18, p = .05) but not with the other amino acids. There were no statistically significant correlations between ATLR and hepatic pmTOR/mTOR, measures of hepatic mitochondrial function determined by Seahorse, or circulating levels of FGF21, insulin or IGF-1 (Supplementary Table 2). Discussion Many observational studies in human populations have found an association between various diet constituents and LTL. Consumption of antioxidant rich, plant-derived foods, and the Mediterranean diet are linked with longer LTL, while shorter LTL have been associated with dietary fats, refined cereals, meat products, and sugar-sweetened beverages (reviewed in refs. (7,16,17)). In a prospective study of young humans, change in telomere length over time was inversely associated with total energy intake as well as that of each macronutrient (18). A number of environmental and lifestyle factors including diet appear to influence telomere length, which may provide a mechanism for their effects on aging and age-related health (7,16). There have been fewer studies of nutrition and telomeres in mice and rats. Telomeres are longer and shorten more rapidly with ageing in rodents than in humans (19,20). Caloric restriction in mice was associated with longer LTL at 15 months (21) while 40% protein restriction maintained telomere length in livers of rats aged 16 months, compared with a decrease in telomere length in those rats on standard chow (20). In our study, in which mice had ad libitum access to food, telomere length assessed by ATLR was positively correlated with carbohydrate intake, and negatively correlated with protein intake, while fat and total energy intake had no effect. Analysis using the Geometric Framework showed that ATLR positively correlated with carbohydrate intake, and highest ATLR was achieved in mice maintained on low protein, high carbohydrate, low fat diets that were ad libitum-fed over a lifetime. The surface generated by Geometric Framework analysis reveals that ATLR doubled from approximately 6 in mice on high protein, low carbohydrate diets to about 13 in those on low protein high, carbohydrate diets. These values are approximately equivalent to 16 and 27 kbp (using an empiric conversion equation (11)), which are similar to values of 19 and 23 kbp seen in the livers of 16-month-old rats maintained on standard chow or protein restriction respectively (20). In the mouse caloric restriction study (21), liver telomere length was not reported, however, caloric restriction was associated with longer telomeres in kidney (94 kbp vs 62 kbp), lung (55 vs 21 kbp), and muscle (14 vs 11 kbp) at 15 months of age. Together, the data in humans and rodents are consistent with a robust effect of nutrition on telomere length in older age, thereby providing a plausible mechanistic link for the effects of nutrition on ageing and age-related health. Telomere length provides a biomarker and a mechanism for aging. The ability of telomere length to predict aging and life span has been reported both in aged mice (19,22,23) and humans (8,9). This relationship has mostly been studied using LTL (6). However, one study in humans found that telomere length in the liver was 12.9 kbp in newborns compared to 8.3 kbp in a centenarian (24), while there is a reduction in the percentage of longer telomeres in livers of rats by 15 months of age (22). In our study, we were not able to directly compare telomere length and life span in the same mice, because the telomeres were evaluated in the livers of mice sacrificed at 15 months of age. However, we were able to compare the responses of both ATLR and median life span to macronutrients in mice from the same experimental cohort. As shown in Figure 2, the surfaces demonstrating the relationship between macronutrients and median life span versus ATLR are almost identical. Mice maintained on low protein, high carbohydrate diets had the longest median life span and the longest ATLR. Our results provide indirect evidence that is consistent with the concept that long telomere length is associated with longer life span. In addition, the rankings by diet of the median life span were compared to the telomere length, and there was a significant positive relationship, such that diets associated with longest median life span were also associated with longest telomeres (Supplementary Figure 2). We then explored whether the association between telomeres, nutrition, and life span is mediated by several mechanisms that are thought to link nutrition with aging (25). There was no association with ATLR and circulating levels of insulin, FGF21 or IGF1, hepatic mTOR activation, nor hepatic mitochondrial activity, although this may reflect the power of the study. However, we did find an association between ATLR and blood levels of amino acids including asparagine, glutamate, and taurine. Previously it has been shown that the chronic circulating levels of most amino acids (except branched chain amino acids) are inversely correlated with protein intake (3). Therefore, the positive association between some amino acids and ATLR in this study may simply reflect the effects of a low protein diet, although only glutamate remained significant after correction for multiple comparisons. On the other hand, taurine is an antioxidant which could influence telomere shortening (26). There are few reports linking amino acids with telomere length. One report in humans studied the relationship between LTL and several circulating amino acids (alanine, glycine, histidine, phenylalanine, leucine, isoleucine, valine, and tyrosine) and found an inverse relationship with phenylalanine (27). In conclusion, mice maintained on low protein, high carbohydrate diets had hepatic longer telomeres which correlated with a longer median life span. Nutrition has a powerful impact on aging and age-related health, which might be mediated in part by its effects on telomere length. Supplementary Material Supplementary data is available at The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online. Funding Funding was from the Australian National Health and Medical Research Council (Project grant 571328, post-doctoral fellowship to S.S.-B.), the Ageing and Alzheimers Institute and the Sydney Medical School Foundation (post-graduate scholarship to R.G.). D.W. is partly supported by the American Australian Association Education Fund. Conflict of Interest None reported. References 1. Mercken EM, Carboneau BA, Krzysik-Walker SM, de Cabo R. Of mice and men: the benefits of caloric restriction, exercise, and mimetics. 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The Journals of Gerontology Series A: Biomedical Sciences and Medical SciencesOxford University Press

Published: Apr 1, 2018

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