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Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats
EXG-09352; No of Pages 10 Experimental Gerontology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Lin Zhao a,d,1, Xuan Zou b,d,1, Zhihui Feng a,d,⁎, Cheng Luo a,d, Jing Liu a,d, Hao Li a,d, Liao Chang a,d, Hui Wang a,d, Yuan Li a,d, Jiangang Long a,d, Feng Gao c,⁎, Jiangang Liu a,d a Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, China b Center for Translational Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, China c Department of Physiology, Fourth Military Medical University, Xi'an, China d Frontier Institute of Life Science, (FIST), Xi'an Jiaotong University, Xi'an, China
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Article history: Received 18 September 2013 Received in revised form 27 January 2014 Accepted 1 February 2014 Available online xxxx
a b s t r a c t
Adipogenesis and lipid accumulation during aging have a great impact on the aging process and the pathogenesis of chronic, age-related diseases. However, little is known about the age-related molecular changes in lipid accumulation and the mechanisms underlying them. Here, using 5-month- and 25-month-old rats (young and old, respectively), we found that epididymal fat is the only tissue to accumulate during aging. By testing tissues rich with mitochondria in old and young animals, we found that the old animals had elevated levels of triglycerides in their muscle, heart and liver tissues but not in their kidneys, while, the mRNA level of fatty acid synthase remained unchanged among the four tissues. Regarding lipid degradation, we determined that the activities of mitochondrial ETC. complexes changed in aged rats (muscle: decreased complex I and V activities; heart: decreased complex I activity; liver: increased complex I and III activities; kidney: decreased complex I and increased complex II activities), while changes in mitochondrial content were not observed in the muscle, heart nor in the liver tissue except increased complex IV and V subunits in aged kidneys. Furthermore, decreased mitochondrial fusion marker Mfn2 and decreased PGC-1α level were observed in the aged muscle, heart and liver but remained unchanged in the kidneys. Down-regulation of Mfn2 with siRNA in 293T cells induced significant mitochondrial dysfunction including decreased oxygen consumption, decreased ATP production, and increased ROS production, followed by increased triglyceride content suggesting a contributing role of decreased mitochondrial fusion to lipid deposit. Meanwhile, judging from autophagy marker p62/SQSTM1 and LC3-II, autophagy was suppressed in the aged muscle, heart and liver but remained unchanged in the kidneys. Taken together, these data suggest that reduction in PGC-1α expression and disruption of mitochondrial dynamics and autophagy might contribute to lipid accumulation during aging. © 2014 Published by Elsevier Inc.
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Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats
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1. Introduction
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Fat tissue is involved with nutrient storage, endocrine function and immunity and undergoes renewal throughout the lifespan of an adult. As the largest organ in humans, fat tissue is thought to play a role in longevity, the progression of age-related disease, inflammation, and metabolic dysfunction (Tchkonia et al., 2010). Total adiposity is usually found to increase during an adult's lifespan. For example, compared
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Keywords: Triglycerides PGC-1α Mitochondrial fusion Autophagy Aging
Abbreviations: SREBP1, Sterol regulatory element-binding protein-1; FAS, Fatty acid synthase; CPT1A, Carnitine palmitoyltransferase IA; CPT1B, Carnitine palmitoyltransferase IB; TG, Triglyceride. ⁎ Corresponding author at: Center for Mitochondrial Biology and Medicine, Xi'an Jiaotong University School of Life Science and Technology, 28 W. Xian-ning Road, Xi'an 710049, China. Tel.: +86 29 82664232. E-mail addresses: [email protected] (Z. Feng), [email protected] (F. Gao). 1 These authors contributed equally to this paper.
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with young adult Caucasian men, older men were found to have increased adiposity according to measurements of body mass index or total body fat mass (Couillard et al., 2000). In middle-aged women, adipose tissue accumulation is increased and contributes to the deterioration of cardiovascular disease risk profiles (Pascot et al., 1999). Generally, adipose tissue is located beneath the skin and around vital organs. During aging, there is an age-related decline in subcutaneous adipose depot size but very late decline in visceral depot size. The age-related loss of subcutaneous fat is accompanied by increased accumulation of fat in the bone marrow, muscle, liver, and other ectopic sites (Cartwright et al., 2007; Kuk et al., 2009). These changes in fat deposition are associated with health risks; it has been shown that excessive fat deposits in the liver increase the incidence of diabetes and cardiovascular disease in middle-aged, non-diabetic subjects (Gastaldelli et al., 2009). The realization that lipid accumulation contributes to the development of health risk factors indicates the importance of lipid
0531-5565/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.exger.2014.02.001
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Cytochrome c, coenzyme Q1, NADP+, antimycin A and dithiothreitol were purchased from Sigma Chemical Co. (St. Louis, MO); Tris base and NADH from Amersco, Inc. (Palm Harbor, FL); 2,6-dichlorophenol indophenol (DCPIP) from Merck & Co., Inc.; rotenone from Riedel De Haen Seelze (Hannover, Germany); antibodies to CPT1A, CPT1B, LC3, Mfn-1, Mfn-2, Drp-1, OPA-1 and PGC-1α from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GAPDH from Cell Signaling Technology (Danvers,
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Sprague–Dawley (SD) male rats were purchased from a commercial breeder (SLAC, Shanghai). The rats were housed in a temperature(22–28 °C) and humidity- (60%) controlled animal room and maintained on a 12-h light/12-h dark cycle (light on from 08:00 a.m. to 08:00 p.m.) with free access to food and water throughout the experiments. Four-week-old male rats weighing 180–200 g were used to start the experiments. After reaching 25 months and 5 months of age (old and young groups, respectively), the animals were sacrificed and various tissue samples were collected and weighed.
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2.3. Isolation of muscle, heart and kidney mitochondria
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At the time of sacrifice and after the tissue weight measurements were taken, the bulk of the skeletal muscle, heart and kidney tissues was collected and frozen in liquid N2. A small portion of fresh tissue from each organ was used to isolate mitochondria as previously described (Shen et al., 2008). Briefly, tissues were trimmed of fat and connective tissue, chopped finely with a pair of scissors, and rinsed in ice-cold medium A (120 mM NaCl, 20 mM HEPES, 2 mM MgCl2, 1 mM EGTA, and 5 g/l bovine serum albumin; pH 7.4) to remove any residual blood. The chopped tissues were resuspended in medium A and homogenized with a hand-held borosilicate glass homogenizer. The homogenate was centrifuged at 600 g for 10 min at 4 °C. The supernatant fluid was subsequently recentrifuged at 17,000 g for 10 min at 4 °C. The pellet containing the mitochondria was resuspended in medium A and then centrifuged at 7000 g for 10 min at 4 °C. The pellet obtained after the last centrifugation was resuspended in medium B (300 mM sucrose, 2 mM HEPES, 0.1 mM EGTA; pH 7.4) and recentrifuged (3500 g, 10 min, 4 °C). The resulting pellet, which contained skeletal muscle, heart or kidney mitochondria, was suspended in a small volume of medium B and stored at −70 °C.
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2.4. Isolation of liver mitochondria
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At the time of sacrifice and after the liver weight measurements were taken, the bulk of the liver was collected and frozen in liquid N2. A small fresh portion was used to isolate mitochondria as previously described (Sun et al., 2010). Briefly, the tissues were rinsed with saline, weighed, and put into an ice-cold isolation buffer containing 0.25 M sucrose, 10 mM Tris, and 0.5 mM EDTA at pH 7.4. The tissues were minced by careful shearing, rinsed to remove residual blood, and then homogenized in the isolation buffer. The homogenate was centrifuged at 1000 g for 10 min; the supernatant was then centrifuged at 10,000 g for 10 min. The mitochondrial pellet was collected and washed twice and resuspended in the isolation buffer. The mitochondrial protein concentrations were determined using a BCA Protein Assay kit (Pierce, IL). The pellets were stored at − 70 °C. All of the operations were carried out at 4 °C.
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2.5. Assays for mitochondrial complex activities
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NADH–ubiquinone reductase (complex I), succinate–CoQ oxidoreductase (complex II), ubiquinol cytochrome c reductase (complex III), cytochrome c oxidase (complex IV) and Mg2 +–ATPase (complex V) were measured spectrometrically using conventional assays as described (Long et al., 2006; Sun et al., 2006).
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MA); and antibodies to complexes I (NDUFS3), II (subunit 30 kDa), III (subunit core 2), IV (subunit I), and V (subunit alpha) from Invitrogen (Carlsbad, CA). Other chemicals and reagents were purchased from Sigma if not otherwise indicated.
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metabolism throughout human life. By targeting lipid metabolism with interventions like exercise (DiPietro, 2010; Rosa et al., 2005) or caloric restriction (Hansen, 2001), age-associated metabolic status and physical function can be improved. Although studies of lipid metabolism have been carried out for decades and several processes have been reported to be involved in its regulation, the mechanisms underlying lipid accumulation during aging are still poorly understood. Lipid homeostasis is normally controlled by sterol regulatory element-binding proteins (SREBPs). The SREBPs transcriptionally activate an enzyme cascade required for the synthesis of endogenous cholesterol, fatty acids, triglycerides (TGs) and phospholipids (Eberle et al., 2004). It has been reported that the SREBP-1mediated regulation of lipogenesis is highly involved in the development of fatty livers (Yahagi et al., 2002) and may also be responsible for lipid accumulation in the muscle (Ikeda et al., 2002; Nadeau et al., 2006). Regarding lipid degradation, nutrient depletion leads to the mobilization of cellular lipid stores to supply free fatty acids for energy, suggesting that there are regulatory and functional similarities between autophagy and lipid metabolism (Singh, 2010; Singh et al., 2009). Inhibition of autophagy decreases triglyceride breakdown, leading to increases in triglyceride levels and lipid droplets in vitro and in vivo (Singh et al., 2009). Genetic inhibition of autophagy in mammalian tissues may induce degenerative changes resembling those associated with aging, and normal and pathological aging is often associated with a reduction in autophagic potential (Rubinsztein et al., 2011). However, aging is a complex process, and the relationship between autophagy and lipid metabolism during aging requires further study. The mitochondria are the organelles that provide the cell with energy and are involved in several diseases and the aging process. The dynamic character of these organelles involves mitochondrial biogenesis and frequent fusion and fission events (Kowald and Kirkwood, 2011). It has been widely accepted that PGC-1α stimulates the efficient induction of NRF-1 and NRF-2 gene expression and binds the promoter of mitochondrial transcription factor A (mtTFA) to regulate mitochondrial biogenesis and fatty acid oxidation (Vega et al., 2000; Wu et al., 1999). While the study of mitochondrial dynamics has received attention in recent years, the mechanisms underlying mitochondrial fusion and fission are still poorly understood. Thus far, studies have suggested that Fis1 and Drp1 are involved in the mitochondrial fission machinery, and OPA, Mfn1, and Mfn2 contribute to the regulation of mitochondrial fusion (Chen and Chan, 2005; Song et al., 2009). The disruption of mitochondrial fusion by the knockdown of mitofusins (Mfns) or OPA1 leads to mitochondrial fragmentation and the accumulation of TGs in adipocytes, suggesting a close connection between the regulation of mitochondrial dynamics and lipid metabolism (Kita et al., 2009). The effect that the remodeling of mitochondrial dynamics may have on lipid metabolism during aging is poorly understood. Therefore, in our present study, we focused on tissues rich in mitochondria—including muscle, heart, liver and kidney tissues—and determined that TGs accumulate in aged muscle, heart and liver tissues but not in the kidneys; this accumulation was accompanied by autophagy inactivation, mitochondrial dynamic alteration and a decrease in PGC-1α expression. Therefore, we propose that mitochondrial metabolism and autophagy might all be involved in lipid accumulation during aging.
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(Sigma). ATP is consumed and light is emitted when firefly luciferase 235 catalyzes the oxidation of D-luciferin. 236
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Total RNA was extracted from 30 mg of tissue using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Two micrograms of RNA was reverse transcribed into cDNA. Quantitative PCR was performed using a real-time PCR system (Eppendorf, Germany). The reactions were performed with SYBR-Green Master Mix (TaKaRa, Dalian, China) using gene-specific primers. The primer sequences were as follows: SREBP1: 5-CGCTACCGTTCCTCTATC-3 (forward) and 5-GGTCTTTCAG TGATTTGC-3 (reverse); FAS: 5-TCCTGTTATCACCCGACT-3 (forward) and 5-GAATACGACCACGCACTAC-3 (reverse); GAPDH: 5-CTCTGCTCCT CCCTGTTCTAGAG-3 (forward) and 5-CAGCCTTGACTGTGCCGTTG-3 (reverse); CPT1A: 5-GACACCAACCCCAACATCCC-3 (forward) and 5TGGATGAAGGCATCGGGACT-3 (reverse); CPT1B: 5-ACCTCTGGGAGTTC GTCCTG-3 (forward) and 5-GGCCTTGGCTACTTGGTACG-3 (reverse). All data were normalized to the expression of the housekeeping gene GAPDH and expressed as relative values using the 2− CT method (Livak and Schmittgen, 2001).
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The samples were lysed with Western and IP lysis buffer (Beyotime, China). The lysates were homogenized, and the homogenates were centrifuged at 13,000 g for 15 min at 4 °C. The supernatants were collected, and the protein concentrations were determined with a BCA Protein Assay kit (Pierce, IL). Equal aliquots (20 μg) of the protein samples were applied to 10% SDS-PAGE gels, transferred to pure nitrocellulose membranes (PerkinElmer Life Sciences, Boston, MA), and blocked with 5% non-fat milk TBST buffer. The membranes were incubated with anti-Mfn1, anti-Mfn2 (1:1000), anti-Drp1 (1:1000), anti-OPA1 (1:1000), anti-PGC-1 (1:1000), anti-GAPDH (1:1000), anti-LC3 (1:1000), anti-CPT1A (1:1000), anti-CPT1B (1:1000) or anti-complex I, II, III, IV or V (1:10,000) at 4 °C overnight. Then, the membranes were incubated with anti-rabbit or anti-mouse antibodies at room temperature for 1 h. Chemiluminescent detection was performed by an ECL Western blotting detection kit (Pierce, IL).
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2′,7′-dichlorodihydrofluorescein diacetate (2′,7′-dichlorofluorescin diacetate; H2DCFDA) is a fluorogenic freely permeable tracer specific for ROS assessment. After treatment, cells were incubated with 10 mM H2DCFDA for 30 min and then washed with PBS three times. Cell lysis was prepared with lysis solution (10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, and 0.5% Triton X-100, pH 7.5). The supernatant (200 μl) was analyzed using a spectrofluorometer with ex 485 nm and em 538 nm (Fluoroskan Ascent, Thermo Fisher Scientific Inc. Waltham, MA). An aliquot of supernatant was used for BCA Protein Assay to determine the concentration of total protein. ROS levels were expressed as relative DCF fluorescence per microgram of protein.
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Cells were cultured in 6-well plates. After treatment, cells were lysed by 0.5% Triton X-100, in 100 mM glycine buffer, pH 7.4. Intracellular ATP level assays were carried out with an ATP bioluminescent assay kit
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All data are reported as the means ± S.E.M. The statistical analysis 243 was performed using t-test analysis. In all comparisons, the level of 244 significance was set at p b 0.05. 245 3. Results
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3.1. Fat accumulation during aging
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The ratios of the weights of specific organ tissues to the whole animal body weight were determined. The ratios of the weights of the heart, liver, bone, muscle, brain, spleen and kidney to the whole body weight were significantly decreased in aging animals; only the ratio of the epididymal fat tissue to the whole body weight was increased (Fig. 1).
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3.2. TG changes in mitochondria-rich tissues during aging
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We then analyzed the TG contents in mitochondria-rich tissues, including muscle, heart, liver and kidney. The TG contents were significantly increased in the muscles, hearts, and livers of the old rats; however, no change was observed in the aged kidneys (Fig. 2).
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3.3. Changes in expression levels of lipogenesis- and beta-oxidation-related 259 genes during aging 260 TG content is closely related to the status of lipogenesis and beta-oxidation. Therefore, tissue RNA and proteins were isolated, and lipogenesis- and beta-oxidation-related markers were analyzed. No significant changes in the mRNA contents of SREBP1 and FAS between the young and old rat tissues were found, except that SREBP1 mRNA was increased in the hearts of old rats (Fig. 3A, B). The increase of SREBP1 mRNA in the heart didn't induce significant protein expression (Fig. S1), as well as other two target genes ACC1 and SCD1 (Fig. S2). Among beta-oxidation-related genes, both mRNA and protein
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293T cells were transfected with Mfn2 siRNA for 48 h. Oxygen consumption rates (OCR) were investigated using Seahorse Extracellular Flux Analyzer (Seahorse Bioscience, Boston, MA, USA) according to the manufacturer's instructions.
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Small portions of the tissues were collected and homogenized in icecold phosphate-buffered saline (PBS). After centrifugation (1000 g, 10 min), the supernatants were collected for triglyceride analysis using commercial clinical diagnosis kits according to the manufacturer's standards and protocols (Jiancheng, Nanjing, China).
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Fig. 1. Accumulation of fat during aging. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected and weighed. The ratios of tissues to body weight were analyzed. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01 as calculated by Student's t-test.
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Fig. 2. Triglyceride (TG) contents in mitochondria-rich tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the skeletal muscle, heart, liver and kidney tissues were collected and analyzed for their TG contents. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
Fig. 3. Lipogenesis and beta-oxidation in the muscles, hearts, livers and kidneys of young and old animals. SD rats aged five months (young) and 25 months (old) were sacrificed, and the skeletal muscle, heart, liver and kidney tissues were collected and total RNA and proteins were prepared for analysis. (A) SREBP1 mRNA, (B) Fas mRNA, (C) CPT1A protein expression, (D) CPT1A statistical analysis, (E) CPT1A mRNA, (F) CPT1B protein expression, (G) CPT1B statistical analysis, and (H) CPT1B mRNA. Values are the means ± S.E.M. from 8 rats, *p b 0.05, **p b 0.01, as calculated by Student's t-test.
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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3.5. Decreases in mitochondrial biogenesis during aging
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Alterations in mitochondrial electron transport chain complex activities are closely related to changes in mitochondrial content. Therefore, we measured mitochondrial biogenesis in the young and aged tissues. We found that PGC-1α, a key regulator of mitochondrial biogenesis, was decreased in the muscle, heart and liver (Fig. 5A, B, and C), while no changes in mitochondrial complex subunit levels were found in these tissues. In the kidneys, PGC-1α expression did not differ between the young and old animals, while complex IV and V subunit levels were found to be significantly elevated in the old animals (Fig. 5D).
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3.6. Alteration of mitochondrial dynamics during aging
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In aged muscle tissue, we found the levels of the mitochondrial fission protein Drp1 to be significantly increased, and mitochondrial
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3.8. Alteration of autophagy during aging
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Autophagy is a catabolic process involving lysosomal degradation of a cell's own components. It is a tightly regulated process that helps to balance the synthesis, degradation and recycling of cellular products. Previous studies have indicated that the accumulation of p62/SQSTM1 and decrease of LC3 II content are signs of autophagy disruption (Larsen et al., 2010; Pankiv et al., 2007; Rusten and Stenmark, 2010). We measured p62/SQSTM1 and LC3 protein levels in four tissue types and found that p62/SQSTM1 was significantly increased in aged muscle, heart, and liver but was unchanged in the kidney (Fig. 8A, B), and LC3-II was significantly decreased in aged muscle, heart, and liver but was unchanged in kidney (Fig. 8C, D). This result is quite similar to the status of mitochondrial dynamics in aged tissues, suggesting a potential
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To further confirm the involvement of mitochondrial dynamic in lipid metabolism we down-regulated Mfn2 with specific siRNA in 293T cells, the efficiency of knockdown was confirmed by measuring protein expression of Mfn2 (Fig. 7A, B). The knockdown of Mfn2 clearly suppressed oxygen consumption (Fig. 7C), decreased ATP content (Fig. 7D), and increased ROS production (Fig. 7E), all of which suggested the mitochondrial dysfunction. As a result, cellular triglycerides were increased after Mfn2 knockdown (Fig. 7F).
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Because the mitochondria are important organelles for energy production and fatty acid oxidation, we isolated them from the aged animal tissues and evaluated their ETC. complex activities. The changes we observed were as follows: complex I and V activities were decreased in the aged muscle mitochondria (Fig. 4A); complex I activity was significantly decreased in the aged heart mitochondria (Fig. 4B); complex I and III activities were significantly increased in the aged liver mitochondria (Fig. 4C); and complex I activity was decreased, while complex II activity was significantly increased, in the aged kidney mitochondria (Fig. 4D).
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fusion proteins Mfn1 and Mfn2 were significantly decreased, while OPA1 levels remained unchanged (Fig. 6A). In aged heart tissue, both Mfn1 and Mfn2 levels were found to be reduced, while Drp1 and OPA1 levels did not change (Fig. 6B). In aged liver tissue, OPA1 and Mfn2 levels were decreased, while Drp1 and Mfn1 levels were unchanged (Fig. 6C). In contrast to the changes observed in these three tissue types, all of the markers of mitochondrial dynamics remained unchanged in aged kidney tissue (Fig. 6D).
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expression levels of CPT1A were found to be significantly increased in the heart, liver, and kidney of old animal, while CPT1B mRNA was found to be increased only in the muscle and heart tissues, and CPT1B protein was found to be increased in the muscle, heart, and kidney tissues (Fig. 3C–H).
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Fig. 4. Electron transport chain (ETC.) complex activities in young and old rat tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected. The mitochondria were isolated from the skeletal muscle, heart, liver and kidney tissues. ETC. complexes I, II, III, IV, and V were analyzed. (A) Muscle, (B) heart, (C) liver, and (D) kidney. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Fig. 5. Mitochondrial biogenesis in young and old rat tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected. The proteins were isolated from the skeletal muscle, heart, liver and kidney tissues. The proteins involved in mitochondrial biogenesis were analyzed by Western blot: (A) muscle, (B) heart, (C) liver, and (D) kidney. Top: Western blot image; Bottom: statistical results. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
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Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Fig. 6. Alteration of mitochondrial dynamics in aged tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected. The proteins were isolated from the skeletal muscle, heart, liver and kidney tissues. The proteins involved in mitochondrial dynamics were analyzed by Western blot. (A) Muscle, (B) heart, (C) liver, and (D) kidney. Left: Western blot image; Right: statistical results. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
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Fig. 7. Down-regulation of Mfn2 impairs mitochondrial function and increases triglyceride content. 293T cells were transfected with Mfn2 siRNA for 48 h, Mfn2 expression was confirmed by Western blot (A: Western blot image, B: statistical analysis). Oxygen consumption (C), cellular ATP content (D), cellular ROS content (E), and cellular triglyceride content (F) were also analyzed. The values are the means ± S.E.M. from three independent experiments. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
connection between autophagy and the remodeling of mitochondrial dynamics.
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Aging is characterized by a progressive decline in cellular function that eventually results in death as cells lose their capacity to respond successfully to injury (Anantharaju et al., 2002). It has been proposed that lipid metabolism may be a key player in the ability to survive to extreme old age (Puca et al., 2008). In this study, we used rats 5 and 25 months of age (young and old, respectively). By determining the ratios of the muscle, heart, liver, kidney and fat tissue weights to whole body weights, we found that the weight ratios of all of these tissues decreased during aging, except for that of fat, which increased. The observation that fat accumulates during aging has been previously reported (Couillard et al., 2000; Pascot et al., 1999). Our results are consistent with and support these previous findings. Past studies have also indicated that during aging lipids are redistributed and accumulate in other tissues, such as skeletal muscle and the liver (Honma et al., 2011; Kim et al., 2009). To more specifically assess this, we collected the mitochondria-rich tissues, including skeletal muscle, the heart, liver and kidneys. We found higher TG contents in the aged muscle, heart and liver tissue but not in the kidney tissue. This observation led us to explore the potential mechanism of lipid redistribution during aging. Two well-known processes—lipogenesis and beta-oxidation are closely tied to TG levels, either via lipid synthesis or degradation (Kersten, 2001; Larigauderie et al., 2006). It was recently discovered that lipogenesis is regulated by the transcription factor SREBP1c, following cleavage into its active mature form (Eberle et al., 2004; Tarling et al., 2004), SREBP1c activates its downstream targets and promotes lipogenesis
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(Shin et al., 2007). In our study, we found that lipogenesis was not increased in aged tissues, although SREBP1 mRNA expression increased significantly in aged heart tissue, its target gene was not significantly affected. These results suggest that the SREBP1c precursor may not have been cleaved into its mature, active form. We therefore speculated that the status of lipogenesis remains unchanged in aged muscle, heart, liver and kidney tissues and that lipid synthesis does not seem to contribute to the accumulation of TGs in aged muscle, heart or liver. In contrast to lipid synthesis, lipid degradation via beta-oxidation may play a critical role in TG accumulation. We measured the mRNA expression levels of CPT1A and CPT1B—the protein products of which are enzymes essential for fatty acid oxidation—and found that, in general, they were increased in all four aged tissue types. Although the mRNA levels were increased, it is hard to definitively conclude from this that beta-oxidation is increased during aging. In addition, there have so far been no studies that have shown increased beta-oxidation during aging. Instead, one animal study did find decreased CPT activity in aged mice heart tissue (Odiet et al., 1995). Mitochondrial function is a complex system. In addition to beta-oxidation, it can be affected by many other processes such as mitochondrial ETC. activities, mitochondrial numbers and the status of mitochondrial dynamics. Therefore, we measured mitochondrial ETC. complex activities and found altered activities in all four aged tissue types. These changes have been individually reported in previous studies showing decreased Complex I activity in muscle (Mansouri et al., 2006), heart (Petrosillo et al., 2009) and kidney (O'Toole et al., 2010). It is well-accepted that this decrease could increase hydrogen peroxide production from the mitochondria, potentially contributing to tissue aging. However, the activity changes we observed in liver tissue were not consistent with a past report (Navarro and Boveris, 2004). Instead of decreasing with age, complex I
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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and III activities were previously found to be increased in aged liver. However, we still assume that mitochondrial function was decreased since the ATP content in aged liver was significantly decreased (data not shown). We then estimated mitochondrial content by measuring mitochondrial ETC. subunit levels and found that mitochondrial contents were not changed in aged muscle, heart and liver tissues. In contrast, PGC-1α, the key regulator of mitochondrial biogenesis, was significantly decreased in these three aged tissue types. In the kidney, unlike in the other three tissue types, we found that complex IV and V subunit expression levels were increased, while PGC-1α expression was unchanged. Similar to the PGC-1α results, we found that mitochondrial fusion markers were significantly decreased in the muscle, heart, and liver tissues but were unchanged in the kidney. One study has shown that the inhibition of mitochondrial fission can induce sustained mitochondrial elongation and changes in senescence-associated phenotype. It was also shown that these changes could be suppressed via the reconstitution of mitochondria, suggesting that the mutual opposition of mitochondrial fusion and fission is required for normal cell growth (Lee et al., 2007). Also, the induction of mitochondrial fusion by silencing the fission protein Drp1 causes a decrease in cellular TG accumulation, while the induction of mitochondrial fission by silencing the fusion proteins Mfn2 and OPA1 causes an increase in this parameter (Kita et al., 2009). Since we observed consistent decrease of Mfn2 in the muscle, heart, and liver, we down-regulated the expression of Mfn2 with siRNA in 293T cells to confirm its contribution to lipid deposit. As expected, Mfn2 knockdown induced significant mitochondrial dysfunction, thereby leading to triglyceride deposit. Therefore, based on our results and those of other studies, we propose that mitochondrial remodeling might also play important role in lipid accumulation during aging. Specifically, decreased mitochondrial fusion might induce
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Fig. 8. Alteration of autophagy in aged tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected. The proteins were isolated from the skeletal muscle, heart, liver and kidney tissues. The alteration of autophagy was analyzed by measuring p62/SQSTM1 (A: Western blot image, B: statistical analysis) and LC3 (C: Western blot image, D: statistical analysis of LC3-II) by Western blot.
impaired mitochondrial function and leads to increased TG accumulation in aged muscle, heart and liver tissues. Despite the fusion/fission cycles, the mitochondria are also more likely to be targeted for autophagy (Tanaka et al., 2010; Twig et al., 2008). When autophagy is triggered, the mitochondria become elongated both in vitro and in vivo, and these elongated mitochondria are spared from autophagic degradation and maintain normal levels of ATP production. Conversely, when elongation is genetically or pharmacologically blocked, the mitochondria consume ATP, precipitating starvation-induced death (Gomes et al., 2011). A study shows that autophagy is disrupted in age-related diseases and in aged tissues (Rubinsztein et al., 2011), and in the current study, we observed decreased LC3-II/LC3-I in aged heart, muscle, and liver expect kidney. Meanwhile, accumulation of p62/SQSTM1 is another sign of autophagy disruption (Larsen et al., 2010; Pankiv et al., 2007; Rusten and Stenmark, 2010), which was found to be increased in the muscle, heart, and kidney expect kidney. Interestingly, autophagy was shown to be altered in aged muscle, heart, and liver, but not in the kidney, which is similar to the mitochondrial dynamic changes we observed. It seems that the alteration of both autophagy and mitochondrial dynamics was observed in the aged tissue types which also have increased TG contents. Taken together, the results from our study provide evidence that the alteration of autophagy and mitochondrial remodeling may contribute to lipid accumulation during aging. However, the mechanistic details and the identification of key regulators will require further study.
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Conflict of interest statement
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The authors have no conflicts of interests.
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Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.exger.2014.02.001.
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Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Lin Zhao a,d,1, Xuan Zou b,d,1, Zhihui Feng a,d,⁎, Cheng Luo a,d, Jing Liu a,d, Hao Li a,d, Liao Chang a,d, Hui Wang a,d, Yuan Li a,d, Jiangang Long a,d, Feng Gao c,⁎, Jiangang Liu a,d a Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, China b Center for Translational Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, China c Department of Physiology, Fourth Military Medical University, Xi'an, China d Frontier Institute of Life Science, (FIST), Xi'an Jiaotong University, Xi'an, China
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Article history: Received 18 September 2013 Received in revised form 27 January 2014 Accepted 1 February 2014 Available online xxxx
a b s t r a c t
Adipogenesis and lipid accumulation during aging have a great impact on the aging process and the pathogenesis of chronic, age-related diseases. However, little is known about the age-related molecular changes in lipid accumulation and the mechanisms underlying them. Here, using 5-month- and 25-month-old rats (young and old, respectively), we found that epididymal fat is the only tissue to accumulate during aging. By testing tissues rich with mitochondria in old and young animals, we found that the old animals had elevated levels of triglycerides in their muscle, heart and liver tissues but not in their kidneys, while, the mRNA level of fatty acid synthase remained unchanged among the four tissues. Regarding lipid degradation, we determined that the activities of mitochondrial ETC. complexes changed in aged rats (muscle: decreased complex I and V activities; heart: decreased complex I activity; liver: increased complex I and III activities; kidney: decreased complex I and increased complex II activities), while changes in mitochondrial content were not observed in the muscle, heart nor in the liver tissue except increased complex IV and V subunits in aged kidneys. Furthermore, decreased mitochondrial fusion marker Mfn2 and decreased PGC-1α level were observed in the aged muscle, heart and liver but remained unchanged in the kidneys. Down-regulation of Mfn2 with siRNA in 293T cells induced significant mitochondrial dysfunction including decreased oxygen consumption, decreased ATP production, and increased ROS production, followed by increased triglyceride content suggesting a contributing role of decreased mitochondrial fusion to lipid deposit. Meanwhile, judging from autophagy marker p62/SQSTM1 and LC3-II, autophagy was suppressed in the aged muscle, heart and liver but remained unchanged in the kidneys. Taken together, these data suggest that reduction in PGC-1α expression and disruption of mitochondrial dynamics and autophagy might contribute to lipid accumulation during aging. © 2014 Published by Elsevier Inc.
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Fat tissue is involved with nutrient storage, endocrine function and immunity and undergoes renewal throughout the lifespan of an adult. As the largest organ in humans, fat tissue is thought to play a role in longevity, the progression of age-related disease, inflammation, and metabolic dysfunction (Tchkonia et al., 2010). Total adiposity is usually found to increase during an adult's lifespan. For example, compared
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Keywords: Triglycerides PGC-1α Mitochondrial fusion Autophagy Aging
Abbreviations: SREBP1, Sterol regulatory element-binding protein-1; FAS, Fatty acid synthase; CPT1A, Carnitine palmitoyltransferase IA; CPT1B, Carnitine palmitoyltransferase IB; TG, Triglyceride. ⁎ Corresponding author at: Center for Mitochondrial Biology and Medicine, Xi'an Jiaotong University School of Life Science and Technology, 28 W. Xian-ning Road, Xi'an 710049, China. Tel.: +86 29 82664232. E-mail addresses: [email protected] (Z. Feng), [email protected] (F. Gao). 1 These authors contributed equally to this paper.
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with young adult Caucasian men, older men were found to have increased adiposity according to measurements of body mass index or total body fat mass (Couillard et al., 2000). In middle-aged women, adipose tissue accumulation is increased and contributes to the deterioration of cardiovascular disease risk profiles (Pascot et al., 1999). Generally, adipose tissue is located beneath the skin and around vital organs. During aging, there is an age-related decline in subcutaneous adipose depot size but very late decline in visceral depot size. The age-related loss of subcutaneous fat is accompanied by increased accumulation of fat in the bone marrow, muscle, liver, and other ectopic sites (Cartwright et al., 2007; Kuk et al., 2009). These changes in fat deposition are associated with health risks; it has been shown that excessive fat deposits in the liver increase the incidence of diabetes and cardiovascular disease in middle-aged, non-diabetic subjects (Gastaldelli et al., 2009). The realization that lipid accumulation contributes to the development of health risk factors indicates the importance of lipid
0531-5565/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.exger.2014.02.001
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Cytochrome c, coenzyme Q1, NADP+, antimycin A and dithiothreitol were purchased from Sigma Chemical Co. (St. Louis, MO); Tris base and NADH from Amersco, Inc. (Palm Harbor, FL); 2,6-dichlorophenol indophenol (DCPIP) from Merck & Co., Inc.; rotenone from Riedel De Haen Seelze (Hannover, Germany); antibodies to CPT1A, CPT1B, LC3, Mfn-1, Mfn-2, Drp-1, OPA-1 and PGC-1α from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GAPDH from Cell Signaling Technology (Danvers,
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Sprague–Dawley (SD) male rats were purchased from a commercial breeder (SLAC, Shanghai). The rats were housed in a temperature(22–28 °C) and humidity- (60%) controlled animal room and maintained on a 12-h light/12-h dark cycle (light on from 08:00 a.m. to 08:00 p.m.) with free access to food and water throughout the experiments. Four-week-old male rats weighing 180–200 g were used to start the experiments. After reaching 25 months and 5 months of age (old and young groups, respectively), the animals were sacrificed and various tissue samples were collected and weighed.
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At the time of sacrifice and after the tissue weight measurements were taken, the bulk of the skeletal muscle, heart and kidney tissues was collected and frozen in liquid N2. A small portion of fresh tissue from each organ was used to isolate mitochondria as previously described (Shen et al., 2008). Briefly, tissues were trimmed of fat and connective tissue, chopped finely with a pair of scissors, and rinsed in ice-cold medium A (120 mM NaCl, 20 mM HEPES, 2 mM MgCl2, 1 mM EGTA, and 5 g/l bovine serum albumin; pH 7.4) to remove any residual blood. The chopped tissues were resuspended in medium A and homogenized with a hand-held borosilicate glass homogenizer. The homogenate was centrifuged at 600 g for 10 min at 4 °C. The supernatant fluid was subsequently recentrifuged at 17,000 g for 10 min at 4 °C. The pellet containing the mitochondria was resuspended in medium A and then centrifuged at 7000 g for 10 min at 4 °C. The pellet obtained after the last centrifugation was resuspended in medium B (300 mM sucrose, 2 mM HEPES, 0.1 mM EGTA; pH 7.4) and recentrifuged (3500 g, 10 min, 4 °C). The resulting pellet, which contained skeletal muscle, heart or kidney mitochondria, was suspended in a small volume of medium B and stored at −70 °C.
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At the time of sacrifice and after the liver weight measurements were taken, the bulk of the liver was collected and frozen in liquid N2. A small fresh portion was used to isolate mitochondria as previously described (Sun et al., 2010). Briefly, the tissues were rinsed with saline, weighed, and put into an ice-cold isolation buffer containing 0.25 M sucrose, 10 mM Tris, and 0.5 mM EDTA at pH 7.4. The tissues were minced by careful shearing, rinsed to remove residual blood, and then homogenized in the isolation buffer. The homogenate was centrifuged at 1000 g for 10 min; the supernatant was then centrifuged at 10,000 g for 10 min. The mitochondrial pellet was collected and washed twice and resuspended in the isolation buffer. The mitochondrial protein concentrations were determined using a BCA Protein Assay kit (Pierce, IL). The pellets were stored at − 70 °C. All of the operations were carried out at 4 °C.
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NADH–ubiquinone reductase (complex I), succinate–CoQ oxidoreductase (complex II), ubiquinol cytochrome c reductase (complex III), cytochrome c oxidase (complex IV) and Mg2 +–ATPase (complex V) were measured spectrometrically using conventional assays as described (Long et al., 2006; Sun et al., 2006).
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MA); and antibodies to complexes I (NDUFS3), II (subunit 30 kDa), III (subunit core 2), IV (subunit I), and V (subunit alpha) from Invitrogen (Carlsbad, CA). Other chemicals and reagents were purchased from Sigma if not otherwise indicated.
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metabolism throughout human life. By targeting lipid metabolism with interventions like exercise (DiPietro, 2010; Rosa et al., 2005) or caloric restriction (Hansen, 2001), age-associated metabolic status and physical function can be improved. Although studies of lipid metabolism have been carried out for decades and several processes have been reported to be involved in its regulation, the mechanisms underlying lipid accumulation during aging are still poorly understood. Lipid homeostasis is normally controlled by sterol regulatory element-binding proteins (SREBPs). The SREBPs transcriptionally activate an enzyme cascade required for the synthesis of endogenous cholesterol, fatty acids, triglycerides (TGs) and phospholipids (Eberle et al., 2004). It has been reported that the SREBP-1mediated regulation of lipogenesis is highly involved in the development of fatty livers (Yahagi et al., 2002) and may also be responsible for lipid accumulation in the muscle (Ikeda et al., 2002; Nadeau et al., 2006). Regarding lipid degradation, nutrient depletion leads to the mobilization of cellular lipid stores to supply free fatty acids for energy, suggesting that there are regulatory and functional similarities between autophagy and lipid metabolism (Singh, 2010; Singh et al., 2009). Inhibition of autophagy decreases triglyceride breakdown, leading to increases in triglyceride levels and lipid droplets in vitro and in vivo (Singh et al., 2009). Genetic inhibition of autophagy in mammalian tissues may induce degenerative changes resembling those associated with aging, and normal and pathological aging is often associated with a reduction in autophagic potential (Rubinsztein et al., 2011). However, aging is a complex process, and the relationship between autophagy and lipid metabolism during aging requires further study. The mitochondria are the organelles that provide the cell with energy and are involved in several diseases and the aging process. The dynamic character of these organelles involves mitochondrial biogenesis and frequent fusion and fission events (Kowald and Kirkwood, 2011). It has been widely accepted that PGC-1α stimulates the efficient induction of NRF-1 and NRF-2 gene expression and binds the promoter of mitochondrial transcription factor A (mtTFA) to regulate mitochondrial biogenesis and fatty acid oxidation (Vega et al., 2000; Wu et al., 1999). While the study of mitochondrial dynamics has received attention in recent years, the mechanisms underlying mitochondrial fusion and fission are still poorly understood. Thus far, studies have suggested that Fis1 and Drp1 are involved in the mitochondrial fission machinery, and OPA, Mfn1, and Mfn2 contribute to the regulation of mitochondrial fusion (Chen and Chan, 2005; Song et al., 2009). The disruption of mitochondrial fusion by the knockdown of mitofusins (Mfns) or OPA1 leads to mitochondrial fragmentation and the accumulation of TGs in adipocytes, suggesting a close connection between the regulation of mitochondrial dynamics and lipid metabolism (Kita et al., 2009). The effect that the remodeling of mitochondrial dynamics may have on lipid metabolism during aging is poorly understood. Therefore, in our present study, we focused on tissues rich in mitochondria—including muscle, heart, liver and kidney tissues—and determined that TGs accumulate in aged muscle, heart and liver tissues but not in the kidneys; this accumulation was accompanied by autophagy inactivation, mitochondrial dynamic alteration and a decrease in PGC-1α expression. Therefore, we propose that mitochondrial metabolism and autophagy might all be involved in lipid accumulation during aging.
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Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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(Sigma). ATP is consumed and light is emitted when firefly luciferase 235 catalyzes the oxidation of D-luciferin. 236
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Total RNA was extracted from 30 mg of tissue using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Two micrograms of RNA was reverse transcribed into cDNA. Quantitative PCR was performed using a real-time PCR system (Eppendorf, Germany). The reactions were performed with SYBR-Green Master Mix (TaKaRa, Dalian, China) using gene-specific primers. The primer sequences were as follows: SREBP1: 5-CGCTACCGTTCCTCTATC-3 (forward) and 5-GGTCTTTCAG TGATTTGC-3 (reverse); FAS: 5-TCCTGTTATCACCCGACT-3 (forward) and 5-GAATACGACCACGCACTAC-3 (reverse); GAPDH: 5-CTCTGCTCCT CCCTGTTCTAGAG-3 (forward) and 5-CAGCCTTGACTGTGCCGTTG-3 (reverse); CPT1A: 5-GACACCAACCCCAACATCCC-3 (forward) and 5TGGATGAAGGCATCGGGACT-3 (reverse); CPT1B: 5-ACCTCTGGGAGTTC GTCCTG-3 (forward) and 5-GGCCTTGGCTACTTGGTACG-3 (reverse). All data were normalized to the expression of the housekeeping gene GAPDH and expressed as relative values using the 2− CT method (Livak and Schmittgen, 2001).
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The samples were lysed with Western and IP lysis buffer (Beyotime, China). The lysates were homogenized, and the homogenates were centrifuged at 13,000 g for 15 min at 4 °C. The supernatants were collected, and the protein concentrations were determined with a BCA Protein Assay kit (Pierce, IL). Equal aliquots (20 μg) of the protein samples were applied to 10% SDS-PAGE gels, transferred to pure nitrocellulose membranes (PerkinElmer Life Sciences, Boston, MA), and blocked with 5% non-fat milk TBST buffer. The membranes were incubated with anti-Mfn1, anti-Mfn2 (1:1000), anti-Drp1 (1:1000), anti-OPA1 (1:1000), anti-PGC-1 (1:1000), anti-GAPDH (1:1000), anti-LC3 (1:1000), anti-CPT1A (1:1000), anti-CPT1B (1:1000) or anti-complex I, II, III, IV or V (1:10,000) at 4 °C overnight. Then, the membranes were incubated with anti-rabbit or anti-mouse antibodies at room temperature for 1 h. Chemiluminescent detection was performed by an ECL Western blotting detection kit (Pierce, IL).
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2′,7′-dichlorodihydrofluorescein diacetate (2′,7′-dichlorofluorescin diacetate; H2DCFDA) is a fluorogenic freely permeable tracer specific for ROS assessment. After treatment, cells were incubated with 10 mM H2DCFDA for 30 min and then washed with PBS three times. Cell lysis was prepared with lysis solution (10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, and 0.5% Triton X-100, pH 7.5). The supernatant (200 μl) was analyzed using a spectrofluorometer with ex 485 nm and em 538 nm (Fluoroskan Ascent, Thermo Fisher Scientific Inc. Waltham, MA). An aliquot of supernatant was used for BCA Protein Assay to determine the concentration of total protein. ROS levels were expressed as relative DCF fluorescence per microgram of protein.
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Cells were cultured in 6-well plates. After treatment, cells were lysed by 0.5% Triton X-100, in 100 mM glycine buffer, pH 7.4. Intracellular ATP level assays were carried out with an ATP bioluminescent assay kit
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All data are reported as the means ± S.E.M. The statistical analysis 243 was performed using t-test analysis. In all comparisons, the level of 244 significance was set at p b 0.05. 245 3. Results
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3.1. Fat accumulation during aging
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The ratios of the weights of specific organ tissues to the whole animal body weight were determined. The ratios of the weights of the heart, liver, bone, muscle, brain, spleen and kidney to the whole body weight were significantly decreased in aging animals; only the ratio of the epididymal fat tissue to the whole body weight was increased (Fig. 1).
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3.2. TG changes in mitochondria-rich tissues during aging
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We then analyzed the TG contents in mitochondria-rich tissues, including muscle, heart, liver and kidney. The TG contents were significantly increased in the muscles, hearts, and livers of the old rats; however, no change was observed in the aged kidneys (Fig. 2).
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3.3. Changes in expression levels of lipogenesis- and beta-oxidation-related 259 genes during aging 260 TG content is closely related to the status of lipogenesis and beta-oxidation. Therefore, tissue RNA and proteins were isolated, and lipogenesis- and beta-oxidation-related markers were analyzed. No significant changes in the mRNA contents of SREBP1 and FAS between the young and old rat tissues were found, except that SREBP1 mRNA was increased in the hearts of old rats (Fig. 3A, B). The increase of SREBP1 mRNA in the heart didn't induce significant protein expression (Fig. S1), as well as other two target genes ACC1 and SCD1 (Fig. S2). Among beta-oxidation-related genes, both mRNA and protein
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293T cells were transfected with Mfn2 siRNA for 48 h. Oxygen consumption rates (OCR) were investigated using Seahorse Extracellular Flux Analyzer (Seahorse Bioscience, Boston, MA, USA) according to the manufacturer's instructions.
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Small portions of the tissues were collected and homogenized in icecold phosphate-buffered saline (PBS). After centrifugation (1000 g, 10 min), the supernatants were collected for triglyceride analysis using commercial clinical diagnosis kits according to the manufacturer's standards and protocols (Jiancheng, Nanjing, China).
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Fig. 1. Accumulation of fat during aging. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected and weighed. The ratios of tissues to body weight were analyzed. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01 as calculated by Student's t-test.
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Fig. 2. Triglyceride (TG) contents in mitochondria-rich tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the skeletal muscle, heart, liver and kidney tissues were collected and analyzed for their TG contents. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
Fig. 3. Lipogenesis and beta-oxidation in the muscles, hearts, livers and kidneys of young and old animals. SD rats aged five months (young) and 25 months (old) were sacrificed, and the skeletal muscle, heart, liver and kidney tissues were collected and total RNA and proteins were prepared for analysis. (A) SREBP1 mRNA, (B) Fas mRNA, (C) CPT1A protein expression, (D) CPT1A statistical analysis, (E) CPT1A mRNA, (F) CPT1B protein expression, (G) CPT1B statistical analysis, and (H) CPT1B mRNA. Values are the means ± S.E.M. from 8 rats, *p b 0.05, **p b 0.01, as calculated by Student's t-test.
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Alterations in mitochondrial electron transport chain complex activities are closely related to changes in mitochondrial content. Therefore, we measured mitochondrial biogenesis in the young and aged tissues. We found that PGC-1α, a key regulator of mitochondrial biogenesis, was decreased in the muscle, heart and liver (Fig. 5A, B, and C), while no changes in mitochondrial complex subunit levels were found in these tissues. In the kidneys, PGC-1α expression did not differ between the young and old animals, while complex IV and V subunit levels were found to be significantly elevated in the old animals (Fig. 5D).
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In aged muscle tissue, we found the levels of the mitochondrial fission protein Drp1 to be significantly increased, and mitochondrial
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3.8. Alteration of autophagy during aging
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Autophagy is a catabolic process involving lysosomal degradation of a cell's own components. It is a tightly regulated process that helps to balance the synthesis, degradation and recycling of cellular products. Previous studies have indicated that the accumulation of p62/SQSTM1 and decrease of LC3 II content are signs of autophagy disruption (Larsen et al., 2010; Pankiv et al., 2007; Rusten and Stenmark, 2010). We measured p62/SQSTM1 and LC3 protein levels in four tissue types and found that p62/SQSTM1 was significantly increased in aged muscle, heart, and liver but was unchanged in the kidney (Fig. 8A, B), and LC3-II was significantly decreased in aged muscle, heart, and liver but was unchanged in kidney (Fig. 8C, D). This result is quite similar to the status of mitochondrial dynamics in aged tissues, suggesting a potential
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To further confirm the involvement of mitochondrial dynamic in lipid metabolism we down-regulated Mfn2 with specific siRNA in 293T cells, the efficiency of knockdown was confirmed by measuring protein expression of Mfn2 (Fig. 7A, B). The knockdown of Mfn2 clearly suppressed oxygen consumption (Fig. 7C), decreased ATP content (Fig. 7D), and increased ROS production (Fig. 7E), all of which suggested the mitochondrial dysfunction. As a result, cellular triglycerides were increased after Mfn2 knockdown (Fig. 7F).
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Because the mitochondria are important organelles for energy production and fatty acid oxidation, we isolated them from the aged animal tissues and evaluated their ETC. complex activities. The changes we observed were as follows: complex I and V activities were decreased in the aged muscle mitochondria (Fig. 4A); complex I activity was significantly decreased in the aged heart mitochondria (Fig. 4B); complex I and III activities were significantly increased in the aged liver mitochondria (Fig. 4C); and complex I activity was decreased, while complex II activity was significantly increased, in the aged kidney mitochondria (Fig. 4D).
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fusion proteins Mfn1 and Mfn2 were significantly decreased, while OPA1 levels remained unchanged (Fig. 6A). In aged heart tissue, both Mfn1 and Mfn2 levels were found to be reduced, while Drp1 and OPA1 levels did not change (Fig. 6B). In aged liver tissue, OPA1 and Mfn2 levels were decreased, while Drp1 and Mfn1 levels were unchanged (Fig. 6C). In contrast to the changes observed in these three tissue types, all of the markers of mitochondrial dynamics remained unchanged in aged kidney tissue (Fig. 6D).
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expression levels of CPT1A were found to be significantly increased in the heart, liver, and kidney of old animal, while CPT1B mRNA was found to be increased only in the muscle and heart tissues, and CPT1B protein was found to be increased in the muscle, heart, and kidney tissues (Fig. 3C–H).
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Fig. 4. Electron transport chain (ETC.) complex activities in young and old rat tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected. The mitochondria were isolated from the skeletal muscle, heart, liver and kidney tissues. ETC. complexes I, II, III, IV, and V were analyzed. (A) Muscle, (B) heart, (C) liver, and (D) kidney. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Fig. 5. Mitochondrial biogenesis in young and old rat tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected. The proteins were isolated from the skeletal muscle, heart, liver and kidney tissues. The proteins involved in mitochondrial biogenesis were analyzed by Western blot: (A) muscle, (B) heart, (C) liver, and (D) kidney. Top: Western blot image; Bottom: statistical results. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
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Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Fig. 6. Alteration of mitochondrial dynamics in aged tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected. The proteins were isolated from the skeletal muscle, heart, liver and kidney tissues. The proteins involved in mitochondrial dynamics were analyzed by Western blot. (A) Muscle, (B) heart, (C) liver, and (D) kidney. Left: Western blot image; Right: statistical results. The values are the means ± S.E.M. from 8 rats per group. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
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Fig. 7. Down-regulation of Mfn2 impairs mitochondrial function and increases triglyceride content. 293T cells were transfected with Mfn2 siRNA for 48 h, Mfn2 expression was confirmed by Western blot (A: Western blot image, B: statistical analysis). Oxygen consumption (C), cellular ATP content (D), cellular ROS content (E), and cellular triglyceride content (F) were also analyzed. The values are the means ± S.E.M. from three independent experiments. *p b 0.05, **p b 0.01, as calculated by Student's t-test.
connection between autophagy and the remodeling of mitochondrial dynamics.
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Aging is characterized by a progressive decline in cellular function that eventually results in death as cells lose their capacity to respond successfully to injury (Anantharaju et al., 2002). It has been proposed that lipid metabolism may be a key player in the ability to survive to extreme old age (Puca et al., 2008). In this study, we used rats 5 and 25 months of age (young and old, respectively). By determining the ratios of the muscle, heart, liver, kidney and fat tissue weights to whole body weights, we found that the weight ratios of all of these tissues decreased during aging, except for that of fat, which increased. The observation that fat accumulates during aging has been previously reported (Couillard et al., 2000; Pascot et al., 1999). Our results are consistent with and support these previous findings. Past studies have also indicated that during aging lipids are redistributed and accumulate in other tissues, such as skeletal muscle and the liver (Honma et al., 2011; Kim et al., 2009). To more specifically assess this, we collected the mitochondria-rich tissues, including skeletal muscle, the heart, liver and kidneys. We found higher TG contents in the aged muscle, heart and liver tissue but not in the kidney tissue. This observation led us to explore the potential mechanism of lipid redistribution during aging. Two well-known processes—lipogenesis and beta-oxidation are closely tied to TG levels, either via lipid synthesis or degradation (Kersten, 2001; Larigauderie et al., 2006). It was recently discovered that lipogenesis is regulated by the transcription factor SREBP1c, following cleavage into its active mature form (Eberle et al., 2004; Tarling et al., 2004), SREBP1c activates its downstream targets and promotes lipogenesis
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(Shin et al., 2007). In our study, we found that lipogenesis was not increased in aged tissues, although SREBP1 mRNA expression increased significantly in aged heart tissue, its target gene was not significantly affected. These results suggest that the SREBP1c precursor may not have been cleaved into its mature, active form. We therefore speculated that the status of lipogenesis remains unchanged in aged muscle, heart, liver and kidney tissues and that lipid synthesis does not seem to contribute to the accumulation of TGs in aged muscle, heart or liver. In contrast to lipid synthesis, lipid degradation via beta-oxidation may play a critical role in TG accumulation. We measured the mRNA expression levels of CPT1A and CPT1B—the protein products of which are enzymes essential for fatty acid oxidation—and found that, in general, they were increased in all four aged tissue types. Although the mRNA levels were increased, it is hard to definitively conclude from this that beta-oxidation is increased during aging. In addition, there have so far been no studies that have shown increased beta-oxidation during aging. Instead, one animal study did find decreased CPT activity in aged mice heart tissue (Odiet et al., 1995). Mitochondrial function is a complex system. In addition to beta-oxidation, it can be affected by many other processes such as mitochondrial ETC. activities, mitochondrial numbers and the status of mitochondrial dynamics. Therefore, we measured mitochondrial ETC. complex activities and found altered activities in all four aged tissue types. These changes have been individually reported in previous studies showing decreased Complex I activity in muscle (Mansouri et al., 2006), heart (Petrosillo et al., 2009) and kidney (O'Toole et al., 2010). It is well-accepted that this decrease could increase hydrogen peroxide production from the mitochondria, potentially contributing to tissue aging. However, the activity changes we observed in liver tissue were not consistent with a past report (Navarro and Boveris, 2004). Instead of decreasing with age, complex I
Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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and III activities were previously found to be increased in aged liver. However, we still assume that mitochondrial function was decreased since the ATP content in aged liver was significantly decreased (data not shown). We then estimated mitochondrial content by measuring mitochondrial ETC. subunit levels and found that mitochondrial contents were not changed in aged muscle, heart and liver tissues. In contrast, PGC-1α, the key regulator of mitochondrial biogenesis, was significantly decreased in these three aged tissue types. In the kidney, unlike in the other three tissue types, we found that complex IV and V subunit expression levels were increased, while PGC-1α expression was unchanged. Similar to the PGC-1α results, we found that mitochondrial fusion markers were significantly decreased in the muscle, heart, and liver tissues but were unchanged in the kidney. One study has shown that the inhibition of mitochondrial fission can induce sustained mitochondrial elongation and changes in senescence-associated phenotype. It was also shown that these changes could be suppressed via the reconstitution of mitochondria, suggesting that the mutual opposition of mitochondrial fusion and fission is required for normal cell growth (Lee et al., 2007). Also, the induction of mitochondrial fusion by silencing the fission protein Drp1 causes a decrease in cellular TG accumulation, while the induction of mitochondrial fission by silencing the fusion proteins Mfn2 and OPA1 causes an increase in this parameter (Kita et al., 2009). Since we observed consistent decrease of Mfn2 in the muscle, heart, and liver, we down-regulated the expression of Mfn2 with siRNA in 293T cells to confirm its contribution to lipid deposit. As expected, Mfn2 knockdown induced significant mitochondrial dysfunction, thereby leading to triglyceride deposit. Therefore, based on our results and those of other studies, we propose that mitochondrial remodeling might also play important role in lipid accumulation during aging. Specifically, decreased mitochondrial fusion might induce
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Fig. 8. Alteration of autophagy in aged tissues. SD rats aged five months (young) and 25 months (old) were sacrificed, and the tissues were collected. The proteins were isolated from the skeletal muscle, heart, liver and kidney tissues. The alteration of autophagy was analyzed by measuring p62/SQSTM1 (A: Western blot image, B: statistical analysis) and LC3 (C: Western blot image, D: statistical analysis of LC3-II) by Western blot.
impaired mitochondrial function and leads to increased TG accumulation in aged muscle, heart and liver tissues. Despite the fusion/fission cycles, the mitochondria are also more likely to be targeted for autophagy (Tanaka et al., 2010; Twig et al., 2008). When autophagy is triggered, the mitochondria become elongated both in vitro and in vivo, and these elongated mitochondria are spared from autophagic degradation and maintain normal levels of ATP production. Conversely, when elongation is genetically or pharmacologically blocked, the mitochondria consume ATP, precipitating starvation-induced death (Gomes et al., 2011). A study shows that autophagy is disrupted in age-related diseases and in aged tissues (Rubinsztein et al., 2011), and in the current study, we observed decreased LC3-II/LC3-I in aged heart, muscle, and liver expect kidney. Meanwhile, accumulation of p62/SQSTM1 is another sign of autophagy disruption (Larsen et al., 2010; Pankiv et al., 2007; Rusten and Stenmark, 2010), which was found to be increased in the muscle, heart, and kidney expect kidney. Interestingly, autophagy was shown to be altered in aged muscle, heart, and liver, but not in the kidney, which is similar to the mitochondrial dynamic changes we observed. It seems that the alteration of both autophagy and mitochondrial dynamics was observed in the aged tissue types which also have increased TG contents. Taken together, the results from our study provide evidence that the alteration of autophagy and mitochondrial remodeling may contribute to lipid accumulation during aging. However, the mechanistic details and the identification of key regulators will require further study.
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Conflict of interest statement
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The authors have no conflicts of interests.
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Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.exger.2014.02.001.
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We thank Dr. Edward Sharman at the University of California, Irvine for reading and editing this manuscript. This work was supported by the National Natural Science Foundation of China (81201023, 30930105) and 985 and 211 Projects of Xi'an Jiaotong University.
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Please cite this article as: Zhao, L., et al., Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.001
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