ARTICLE |
Correspondence to: Alfred Lohninger, Dept. of Medical Chemistry, Währinger Str. 10, A-1090 Vienna, Austria. E-mail: alfred.lohninger@univie.ac.at
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Summary |
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Aging affects oxidative metabolism in liver and other tissues. Carnitine acyltransferases are key enzymes of this process in mitochondria. As previously shown, the rate of transcription and activity of carnitine palmitoyltransferase CPT1 are also related to carnitine levels. In this study we compared the effect of dietary L-carnitine (100 mg L-carnitine/kg body weight/day over 3 months) on liver enzymes of aged rats (months 2124) to adult animals (months 69) and age-related controls for both groups. The transcription rate of CPT1, CPT2, and carnitine acetyltransferase (CRAT) was determined by quantitative reverse transcription real-time PCR (RTQPCR) and compared to the activity of the CPT1A enzyme. The results showed that the transcription rates of CPT1, CPT2, and CRAT were similar in aged and adult control animals. Carnitine-fed old rats had a significant (p<0.05) 812-fold higher mean transcription rate of CPT1 and CRAT compared to aged controls, adult carnitine-fed animals, and adult controls, whereas the transcription rate of CPT2 was stimulated 23-fold in carnitine-fed animals of both age groups. With regard to the enzymatic activity of CPT1 there was a 1.5-fold increase in the old carnitine group compared to all other groups. RNA in situ hybridization also indicated an enhanced expression of CPT1A in hepatocytes from L-carnitine-supplemented animals. These results suggest that L-carnitine stimulates transcription of CPT1, CPT2, and CRAT as well as the enzyme activity of CPT1 in the livers of aged rats. (J Histochem Cytochem 50:205212, 2002)
Key Words: carnitine palmitoyltransferases, (CPT1 and CPT2), carnitine acetyltransferase, (CRAT), transcription rate, enzyme activity, rat liver
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Introduction |
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THE AGING PROCESS is often associated with altered mitochondrial function and decreased energy production by oxidative metabolism (
Mitochondrial energy production from the main oxidative substrates, i.e., fatty acids and glucose, is regulated by carnitine acyltransferases such as CPT1 and carnitine acetyltransferase (CRAT), the latter regulating the level of acetyl CoA, which is a potent physiological inhibitor of the pyruvate dehydrogenase complex (
The best described function of CPT is its role in the carnitine-dependent fatty acid transport system through the inner mitochondrial membrane. This consists of the malonyl-CoA-sensitive CPT1 localized in the outer mitochondrial membrane, carnitine acylcarnitine translocase, an integral inner membrane protein, and carnitine palmitoyltransferase 2 (CPT2), which is localized on the matrix side of the inner membrane (
Various isoforms of CPT1 are also part of the peroxisomal and microsomal acyltransferase system, the latter appearing to influence whether triglycerides accumulate in the cytosol or whether they are exported via lipoproteins (very low-density lipoproteins; VLDLs) to peripheral tissues (
However, it should be mentioned that a complex metabolic equilibrium exists among the various carnitine pools in the different body compartments. In aged rats there is a significant decrease of total carnitine levels in the brain, serum, heart, and skeletal muscle, accompanied by an increase in the liver (
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Materials and Methods |
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Animals
Male SpragueDawley rats (OFA-17; Versuchstierzuchtanstalt der Universität Wien, Himberg), aged 7 months (adult, n=12) and 21 months (old, n=24) were used in this study. They were housed separately in Makrolon-III cages under standard conditions (25C; 5060% atmospheric humidity; light from 06001800 hr; Altromin-R rat diet and tapwater ad libitum). All procedures were performed strictly in accordance with the Austrian law for animal tests and approved by the Austrian Commission for Animal Test Affairs (TVNr:GZ 68.205/31-Pr/4/2000).
Experimental Assay for Validating the Effect of Dietary Carnitine
L-Carnitine was obtained from Lonza (Basel, Switzerland). After overnight (12-hr) water deprivation, animals were weighed and received an L-carnitine solution (60 mg L-carnitine/ml water) to achieve a total dosage of 100 mg/kg body weight/day. After animals had drunk the L-carnitine solution, bottles were filled with water. Controls had drinking water ad libitum. After 3 months the animals were sacrificed.
Briefly, after application of narcosis with a mixture of oxygen:nitrous oxide (1:2) and 4% halothane per inhalation, the thorax was opened. Animals were exsanguinated by withdrawal of blood from the right ventricle using sterile heparin- and EDTA Vacuettes. Livers were excised and frozen at -180C.
RNA In Situ Hybridization (RISH)
Ten-µm frozen sections were mounted on poly-L-lysine-coated slides and fixed in 3% phosphate buffered formaldehyde, dehydrated in ethanol, and air-dried. For each specimen, 50 ng of a digoxigenin-labeled PCR product for rat CPT1A was diluted in a commercially available hybridization buffer (Hybrisol IV; Oncor, Gaithersburg, MD). To ensure perfect permeabilization and denaturation of probes and targets, slides were placed in a metal box and heated for 5 min in an 80C water bath and then incubated overnight at 37C. After hybridization, slides were washed in PBS, three times for 10 min and incubated with anti-digoxigenin antibodies conjugated to rhodamine (Roche Diagnostics; Mannheim, Germany) diluted 1:10 in blocking solution (Roche) for 30 min at 37C. Afterwards, slides were washed with PBS (three times for 10 min), rinsed with water, and counterstained with 2 µg/ml DAPI (Sigma; St Louis, MO) in McIlvaine's buffer (0.1 M citric acid monohydrate + 0.2 M disodium hydrogen phosphate) for 20 min at room temperature, rinsed with distilled water, air-dried, and covered with 50% glycerol in PBS. For evaluation of slides a Zeiss Axiophot Microscope in combination with a Filterset 40 (according to D. Pinkel) and the Power Gene 760 Karyotyping & Probe System PSI (Halladale, UK) was used (G65, G63) (
A fluorescein-labeled PCR product for ß-actin was used as positive control probe. As negative controls, hybridization buffer without probe was added to the reference sections.
Quantitative Reverse Transcription Polymerase Chain Reaction (RTQPCR)
For preparation of mRNA, 0.5-cm3 pieces of rat liver were covered with 4 M guanidine isothiocyanate in sterile Petri dishes and minced with sterile scalpels. mRNA preparation and subsequent cDNA preparation were done with commercially available systems (Amersham Pharmacia Biotech Europe; Freiburg, Germany).
RT-PCR was carried out using a Lightcycler System (Roche Diagnostics) that allows amplification and detection (by fluorescence) in the same tube, using a kinetic approach. Lightcycler PCR reactions were set up in microcapillary tubes using 2 µl cDNA (10 ng) with 18 µl of a LightCycler-Fast Start DNA Master Mix SYBR Green I reaction mix (Roche Diagnostics) as described (
For calibration, standards ranging from 10 pg down to 1 fg were prepared from RT-PCR products purified after electrophoresis on 2% agarose gels.
As a negative control, samples with water instead of target cDNA were used.
Evaluation of quantitative RT-PCR was made after converting the amount of target amplified (fg per 20-µl assay containing 10 ng cDNA) to copy numbers. Transcription rate was calculated as number of copies of CPT1, CPT2, or CRAT/100 copies of ß-actin or G6PDH, which were used as genes for standardization (
Determination of CPT1 Activity
CPT1 activity was analyzed as described (
CPT activity was assayed in these supernatants spectrophotometrically by following the release of CoA-SH from palmitoyl-CoA using the general thiol reagent DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)); 850 µl Tris-HClDTNB buffer (116 mM Tris, 2.5 mM EDTA, 2 mM DTNB, 0.2% Triton X-100, pH 8.0). Fifty µl homogenization buffer and 50 µl cleared supernatant was added to four semi-microcuvettes (Greiner). After 5-min preincubation at 30C, 50 µl palmitoyl-CoA (1 mM dissolved in double distilled water) was added to three cuvettes. The fourth cuvette was used as a blank, adding 50 µl water instead of palmitoyl-CoA. The reaction was then started by adding 5 µl L-carnitine solution (1.2 mM dissolved in 1 M Tris, pH 8), immediately followed by photometric measurement (Hitachi U-2010; 30C, 412 nm) for 180 sec (
The protein content of the cleared supernatants was determined according to the method of
Statistics
Statistical comparison between the groups used ANOVA followed by Dunnet's t-test for multiple comparison (
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Results |
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Characteristics of Experimental Animals
Mean body weight of male SpragueDawley rats, aged 21 months, was significantly higher than the body weight of 7-month-old rats (568 g vs 490 g). Oral pretreatment with L-carnitine did not significantly affect body weight. To ensure that experimental animals consumed the entire amount of L-carnitine, animals received an adequate amount of an L-carnitine solution (60 mg L-carnitine/ml water) after overnight (12-hr) water deprivation to achieve a total dosage of 100 mg/kg body weight/day. After animals had consumed the L-carnitine solution completely, bottles were refilled with pure tapwater. Controls received drinking water ad libitum.
Influence of Age and Carnitine Supplementation on Transcription of Carnitine Acyltransferases
Our first goal was to determine whether there is an age-related difference in the transcription rates of liver carnitine acyltransferases similar to those described for CPT1B in muscle and heart and to see how dietary L-carnitine might influence this. Two methods, i.e., RTQPCR and RISH, were used for these investigations.
Results of RTQPCR
As shown in Fig 1 and Table 1, dietary L-carnitine resulted in a 10-fold increase of the mean transcription rate for CPT1 and CRAT and a twofold increase for CPT2 in livers from the carnitine group of old animals in contrast to the age-matched control group. In livers of adult animals, dietary L-carnitine also significantly stimulated the transcription rate of CPT2 but not of CPT1 and CRAT. Furthermore, there were no significant differences in the transcription rates of CPT1, CPT2, and CRAT between old and adult controls.
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For quantitating transcription of CPT1, CPT2, and CRAT, RTQPCR using the LightCycler SYBR Green technique was employed, which has been shown to produce more precise results than other approaches for mRNA quantification such as probe hybridization (Northern blots) or band densitometry (
The level of gene expression was measured by relative or absolute RTQPCR. For absolute quantitation, a standard curve relates the PCR signal to the input copy numbers of a defined template (
Influence of Age and Carnitine Supplementation on Activity of CPT1A
Our second goal was to test if there was an age-related effect of dietary L-carnitine on enzyme activity of CPT1A in rat hepatocytes. As shown in Table 2 and Fig 2, the enzyme activity of CPT1 in aged carnitine-supplemented rats was 1.5-fold higher than in the age-matched control group (p<0.01). Again, there was no significant difference between aged and adult control animals. L-Carnitine supplementation had a slight but not significant stimulating effect on the activity of CPT1A in livers of adult animals. Furthermore, the results displayed in Table 2 show that there was a good correlation between enzyme activity and transcription rate as defined by the CPT1A/ß-actin ratio (p<0.001) or the CPT1A/G6PD ratio (p = 0.01) in old carnitine-treated rats and in age-related controls but not in the corresponding groups of adult animals.
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Results of Fluorescent RISH
The third goal of this study was to test if RISH would be a suitable tool for measuring the effect of L-carnitine supplementation on hepatic tissue. Results of fluorescent RISH of livers from carnitine-supplemented rats compared to controls are shown in Fig 3. Especially in the border zones of cryostat sections of both old and young carnitine-fed animals, more hepatocytes appeared to be positive for CPT1 expression compared to the age-related controls.
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The RISH performed in this study showed a higher density of hybridization signals for CPT1 in the border zones of liver parenchyma in both aged and adult carnitine-supplemented animals compared to unsupplemented controls (Fig 3). This supports a previous report showing a great heterogeneity of CPT activity in various hepatocyte fractions of liver parenchyma (
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Discussion |
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The present study is the first report demonstrating that dietary L-carnitine causes an alteration in the expression of CPT1A, CPT2, and CRAT at the mRNA level and for CPT1A also on the level of enzymatic activity in livers of old rats in contrast to livers from adult animals. This work therefore provides evidence for a significant correlation between altered enzyme activity and transcription rate of CPT1, especially in livers of carnitine-supplemented old animals, but no evidence for a significant age-correlated alteration of the transcription rate and enzyme activity of CPT1A without carnitine supplementation.
The results obtained in liver, where CPT1A is the predominant isoform, were in sharp contrast to findings in heart, skeletal muscle, and adipose tissue ( (peroxisome proliferator-activated receptor alpha), whereas CPT1A has a TRE (thyroid hormone-responsive element) and a CCAAT box, which is frequently observed in glucocorticoid-responsive genes.
This might be caused by different sensitivity to carnitine stimulation due to downregulated thyroid hormones in senescent organisms. (
An age-associated slower mRNA degradation does not appear to be involved because mitochondrial CPT2 was stimulated by dietary L-carnitine independently of age, although previous studies (e.g.,
Furthermore, there was a significant correlation between CPT1A mRNA content and activity, whereby the enhancement of mRNA transcription was more pronounced. It is well established that long-term changes in CPT1 activity are based on changes at the transcriptional level (
Methodological pitfalls, causing the observed differences between transcription rate and activity of CPT, can be mostly excluded for the following reasons. (a) Due to the necessary dilution steps in the in vitro assay of CPT activity, an inhibition of the enzyme by malonyl-CoA is negligible. (b) The affinity of CPT1 for the acyl-CoA substrate alters under ketogenic conditions and phosphorylation as induced by, e.g., glucagon or forskolin (
Comparison of isolated enzyme activities may be misleading in general, since in vitro assays are non-physiological compared to the in vivo environment of the enzyme in the cell. Although other factors such as long-chain fatty acids, clofibrate, the plasticizer DEHP [=2-(diethyl-hexylphthtalate], and glucagon (e.g.,
Further studies on key enzymes of other metabolic pathways may show whether dietary L-carnitine can cause changes in the utilization of fuel in aged organisms.
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Acknowledgments |
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Supported by the Herzfelder Stiftung and the Austrian Federal Chancellery.
Thanks are due to Jennifer Hirsch for expert help with preparation of cryostat sections.
Received for publication May 1, 2001; accepted September 19, 2001.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abo-Hashema KAH, Cake MH, Power GW, Clarke D (1999) Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases. J Biol Chem 274:35577-35582
Bieber LL, Abraham T, Helmrath T (1972) A rapid spectrophotometric assay for carnitine palmitoyltransferase. Anal Biochem 50:509-518[Medline]
Bodenner DL, McClaskey JH, Kim MK, Mixson AJ, Weintraub BD (1992) The proto-oncogenes c-fos and c-jun modulate thyroid hormone inhibition of human thyrotropin beta subunit gene expression in opposite directions. Biochem Biophys Res Commun 189:1050-1056[Medline]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254[Medline]
Brady PS, Brady LJ (1989a) Regulation of carnitine palmitoyltransferase in vivo by glucagon and insulin. Biochem J 258:677-682[Medline]
Brady PS, Brady LJ (1989b) Effects of clofibrate and acetylsalicylic acid on hepatic arniti carnitine palmitoyltransferase synthesis. Biochem Pharmacol 38:811-814[Medline]
Brady LJ, Ramsay RR, Brady PS (1991) Regulation of carnitine acyltransferase synthesis in lean and obese Zucker rats by dehydroepiandrosterone and clofibrate. J Nutr 121:525-531[Medline]
Bremer J, Norum KR (1967) Palmityl-CoA: carnitine O-palmityltransferase in the mitochondrial oxidation of palmityl-CoA. Eur J Biochem 1:427-433[Medline]
Broadway NM, Gooding JM, Saggerson ED (1999) Carnitine acyltransferases and associated transport processes in the endoplasmic reticulum. Missing links in the VLDL story? Adv Exp Med Biol 466:59-67[Medline]
Brown NF, Hill JK, Esser V, Kirkland JL, Corkey BE, Foster DW, McGarry JD (1997) Mouse white adipocytes and 3T3-L1 cells display an anomalous pattern of carnitine palmitoyltransferase (CPT) I isoform expression during differentiation. Inter-tissue and inter-species expression of CPT I and CPT II enzymes. Biochem J 327(pt 1):225-231[Medline]
Chatelain F, Kohl C, Esser V, McGarry JD, Girard J, Pegorier JP (1996) Cyclic AMP and fatty acids increase carnitine palmitoyltransferase I gene transcription in cultured fetal rat hepatocytes. Eur J Biochem 235:789-798[Abstract]
Chiu KM, Schmidt MJ, Havighurst TC, Shug AL, Dayes RA, Keller ET, Gravenstein S (1999) Correlation of serum L-carnitine and dehydro-epiandrosterone sulphate levels with age and sex in healthy adults. Age Ageing 28:211-216[Abstract]
Cook GA (1984) Differences in the sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA are due to differences in Ki values. J Biol Chem 259:12030-12033
Cook GA, Edwards TL, Jansen MS, Bahouth SW, Wilcox HG, Park EA (2001) Differential regulation of carnitine palmitoyltransferase-I gene isoforms (CPT-I alpha and CPT-I beta) in the rat heart. J Mol Cell Cardiol 33:317-329[Medline]
Cook GA, Otto DA, Cornell NW (1980) Differential inhibition of ketogenesis by malonyl-CoA in mitochondria from fed and starved rats. Biochem J 192:955-958[Medline]
Costell M, O'Connor JE, Grisolia S (1989) Age-dependent decrease of carnitine content in muscle of mice and humans. Biochem Biophys Res Commun 161:1135-1143[Medline]
Dunnet CW (1964) New tables for multiple comparisons with a control. Biometrics 20:482-485
Gadaleta MN, Petruzzella V, Fracasso F, FernandezSilva P, Cantatore P (1990) Acetyl-L-carnitine increases cytochrome oxidase subunit I mRNA content in hypothyroid rat liver. FEBS Lett 277:191-193[Medline]
Grantham BD, Zammit VA (1988) Role of carnitine palmitoyltransferase I in the regulation of hepatic ketogenesis during the onset and reversal of chronic diabetes. Biochem J 249:409-414[Medline]
Guzman M, Velasco G, Castro J, Zammit VA (1994) Inhibition of carnitine palmitoyltransferase I by hepatocyte swelling. FEBS Lett 344:239-241[Medline]
Hagen TM, Ingersoll RT, Wehr CM, Lykkesfeldt J, Vinarsky V, Bartholomew JC, Song MH, Ames BN (1998) Acetyl-L-carnitine fed to old rats partially restores mitochondrial function and ambulatory activity. Proc Natl Acad Sci USA 95:9562-9566
Hagen TM, Yowe DL, Bartholomew JC, Wehr CM, Do KL, Park JY, Ames BN (1997) Mitochondrial decay in hepatocytes from old rats: membrane potential declines, heterogeneity and oxidants increase. Proc Natl Acad Sci USA 94:3064-3069
Harano Y, Kashiwagi A, Kojima H, Suzuki M, Hashimoto T, Shigeta Y (1985) Phosphorylation of carnitine palmitoyltransferase and activation by glucagon in isolated rat hepatocytes. FEBS Lett 188:267-272[Medline]
Harrison DC, Medhurst AD, Bond BC, Campbell CA, Davis RP, Philpot KL (2000) The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia. Mol Brain Res 75:143-149[Medline]
Heo K, Lin X, Odle J, Han IK (2000) Kinetics of carnitine palmitoyltransferase I are altered by dietary variables and suggest a metabolic need for supplemental carnitine in young pigs. J Nutr 130:2467-2470
Jansen MS, Cook GA, Song S, Park EA (2000) Thyroid hormone regulates carnitine palmitoyltransferase Ialpha gene expression through elements in the promoter and first intron. J Biol Chem 275:34989-34997
Jin SJ, Hoppel CL, Tserng KY (1992) Incomplete fatty acid oxidation. J Biol Chem 267:119-125
Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H, Herrlich P (1990) Antitumor promotion and anti-inflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62:1189-1204[Medline]
Kerner J, Hoppel C (2000) Fatty acid import into mitochondria. Biochim Biophys Acta 1486:1-17[Medline]
Lohninger A, Krieglsteiner HP, Hajos F, Stangl H, Marz R (1996) Effects of prenatal treatment with betamethasone, L-carnitine, or betamethasone-L-carnitine combinations on the phosphatidylcholine content and composition of the foetal and maternal rat lung. Eur J Clin Chem Clin Biochem 34:387-391[Medline]
Lohninger A, Krieglsteiner HP, Salzer H, Erhardt W, Eppl W, Kaiser E (1986a) Studies on the effects of L-thyroxine, L-carnitine and L-thyroxine-L-carnitine combination on dipalmitoyl phosphatidylcholine content and on phosphatidylcholine species composition of fetal rat lungs. Pediatr Res 20:185-192
Lohninger A, Krieglsteiner HP, Salzer H, VytiskaBinstorfer E, Riedl W, Erhardt W (1986b) Studies on the effects of betamethasone, L-carnitine and bethamethasone-L-carnitine combinations on the dipalmitoyl phosphatidylcholine content and phosphatidylcholine species composition in foetal rat lungs. J Clin Chem Clin Biochem 24:361-368[Medline]
Maccari F, Arseni A, Chiodi P, Ramacci MT, Angelucci L (1990) Levels of carnitines in brain and other tissues of rats of different ages:effect of acetyl-L-carnitine administration. Exp Gerontol 25:127-134[Medline]
Maebashi M, Imamura A, Yoshinaga K (1982) Effect of aging on lipid and carnitine metabolism. Tohoku J Exp Med 138:231-236[Medline]
McGarry JD, Brown NF (1997) The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur J Biochem 244:1-14[Abstract]
Moir AM, Zammit VA (1995) Insulindependent and extremely rapid switch in the partitioning of hepatic fatty acids from oxidation to esterification in starved-refed diabetic rats. Possible roles for changes in cell pH and volume. Biochem J 305:953-958[Medline]
Nowotny H, Karlic H, Grüner H, Hirsch J, Vesely M, Nader A, Heinz R (1996) Cytogenetic findings in 175 patients indicate that items of the Kiel classification should not be disregarded in the REAL classification of lymphoid neoplasms. Ann Hematol 72:291-301[Medline]
Odiet JA, Boerrigter M, Wei JY (1995) Carnitine palmitoyl transferase-I activity in the aging mouse heart. Mech Ageing Dev 79:127-136[Medline]
Saheki T, Li MX, Kobayashi K (2000) Antagonizing effect of AP1 on glucocorticoid induction of urea cycle enzymes: a study of hyperammonemia in carnitine deficient, juvenile visceral steatosis mice. Mol Genet Metab 71:545-551[Medline]
Shigenaga MK, Hagen TM, Ames BN (1994) Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci USA 91:10771-10778
Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW (2000) Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 285:194-204[Medline]
Starritt EC, Howlett RA, Heigenhauser GJ, Spriet LL (2000) Sensitivity of CPT I to malonyl-CoA in trained and untrained human skeletal muscle. Am J Physiol 278:E462-468
Suzuki T, Higgins PJ, Crawford DR (2000) Control selection for RNA quantitation. BioTechniques 29:332-337[Medline]
Thakur MK, Chaurasia P (1997) Nuclease susceptibility of the rat liver satellite DNA-containing chromatin decreases with age. Mol Cell Biochem 171:445-448
van der Leij FR, Huijkman NC, Boomsma C, Kuipers JR, Bartelds B (2000) Genomics of the human carnitine acyltransferase genes. Mol Genet Metab 71:139-153[Medline]
van Leeuwen FW, Fischer DF, Benne R, Hol EM (2000) Molecular misreading. A new type of transcript mutation in gerontology. Ann NY Acad Sci 908:267-281
Velasco G, Gomez del Pulgar T, Carling D, Guzman M (1998) Evidence that the AMP-activated protein kinase stimulates rat liver carnitine palmitoyltransferase I by phosphorylating cytoskeletal components. FEBS Lett 439:317-320[Medline]
Wang ZW, Pan WT, Lee Y, Kakuma T, Zhou YT, Unger RH (2001) The role of leptin resistance in the lipid abnormalities of aging. FASEB J 15:108-114
Winer J, Jung CK, Shackel I, Williams CT (1997) Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270:41-49
Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ (1999) The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. BioTechniques 22:176-181