Acetyl- l -Carnitine Supplementation to Old Rats Partially Reverts the Age-Related Mitochondrial Decay of Soleus Muscle by Activating Peroxisome Proliferator-Activated Receptor γ Coactivator-1α–Dependent Mitochondrial Biogenesis

Effects of aging and ALCAR treatment on soleus mtDNA content

The mtDNA content was evaluated by quantitative real-time PCR (qRT-PCR) as the mtDNA/nuclear DNA ratio in soleus skeletal muscle from three groups of male Fisher 344 (Charles River) rats including young (6 months old), aged (28 months old), and aged rats supplemented daily with ALCAR in drinking water at a dose of 100 mg/kg body weight (for 2 months before sacrifice, performed at 28 months of age).

Food intake and water consumption were not significantly different between the aged groups. Total DNA was prepared from about 40–50 mg of skeletal muscle, as described by Pesce et al. ¹⁵ The measurement of the mtDNA content was carried out using specific primers and TaqMan probes, accurately designed by Primer Express software (Applied Biosystems, Foster City, CA) for the mitochondrial D-loop region (D-loopFor, 5′-GGTTCTTACTTCAGGGCCATA-3′; D-loopProbe, 5′-6FAM-TTGGTTCATCGTCCATACGTTCCCCTTA-TAMRA-3′; D-loopRev, 5′-GATTAGACCCGTTACCATCGAGAT-3′). For the nuclear β-actin gene, primers were: β-actinFor: 5′-GGGATGTTTGCTCCAACCAA-3′; β-actinRev, 5′-GCGCTTTTGACTCAAGGATTTAA-3′; β-actinProbe, 5′-VIC-CGGGTCGCCTTCACCGTTCCAGTT-TAMRA-3′). The method has been validated by primer-limiting experiments to determine the proper primers concentrations (200 nM for each primer pair and 100 nM for both probes) and by evaluating the equal reaction efficiency of the two amplicons. Each sample was analyzed in triplicate, and fluorescence spectra were monitored by the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, CA).

The difference in threshold cycle values (ΔCt, namely Ct D-loop − Ct β-actin) was used as a measure of the relative abundance of the mitochondrial genome. To compare the mtDNA amount between groups of rats of different ages, the younger age animals class was taken as the reference one and the relative amount of mtDNA to nuclear DNA was calculated by the following equation: R = 2^−ΔΔCt, where R is the calculated ratio and ΔΔCt is the ΔCt_{analyzed class} − ΔCt_{reference class} value. Data were submitted to analysis of variance (ANOVA) with Tukey Honestly Significant Difference (HSD) post hoc test using SPSS Base 11.5 software (SPSS Inc., Chicago, IL) to assess the significance of the differences observed between groups.

The changes of the mtDNA content during aging and in response to ALCAR feeding in the soleus skeletal muscle are reported in Table 1. The analysis shows an age-dependent 50% decrease of the mtDNA/nDNA ratio. Very interestingly, the soleus muscle of 28-month-old rats treated with ALCAR for 2 months shows only a 25% decrease of the mtDNA content. Therefore, the ALCAR treatment seems to partially revert the age-dependent reduction of the mtDNA content in soleus skeletal muscle. Such mtDNA depletion has been already reported in aged soleus muscle by other studies ¹⁶ and, more recently, by the authors in the soleus muscle of 28-month-old Wistar rats ¹⁵ and Fisher rats. ¹⁷

Effects of aging and ALCAR treatment on citrate synthase activity

The mitochondrial matrix enzyme citrate synthase (CS) activity was determined as a marker of mitochondrial mass. ¹⁸ Total proteins were extracted from ∼20 mg of soleus muscle by homogenization in buffer (pH 7.4) containing 100 mM mannitol, 1 mM adenosine triphosphate (ATP), 0.2% bovine serum albumin (BSA), 100 mM KCl, 3 mM MgCl₂, 5 mM Tris, and 1 mM EDTA and centrifuged at 700 × g for 10 min at 4°C. Protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA) according to the supplier's instructions. Citrate synthase activity (μmol · min⁻¹ · g⁻¹ muscle) was determined spectropho-tometrically in soleus muscle homogenates according to the method of Srere ¹⁹ and normalized through comparison with the young group value set equal to 1.

The activity (see Table 1) decreased significantly (about 50%) with aging, whereas in 28-month-old rats treated with ALCAR for 2 months, there was only a statistically significant 25% decrease of citrate synthase activity, further suggesting a partial reversal of the age-related mitochondrial decay through ALCAR supplementation.

Effects of aging and ALCAR treatment on the level of some mitochondrial and nuclear transcripts and on the expression of nuclear genes related to mitochondrial biogenesis

qRT-PCR was carried out to obtain data on mitochondrial (COXI, 16S rRNA) and nuclear (COXIV, PGC1-α, mitochondrial transcription factor A [TFAM], mitochondrial transcription factor 2B [TFB2]) transcripts from genes involved, respectively, in mitochondrial functionality and biogenesis in normally fed versus ALCAR-fed aged rats. The relative mRNA abundance was normalized with respect to that of the young control group (set as 1). Total RNA was extracted and reverse-transcribed as described by Cassano et al., ¹¹ whereas real time-PCR amplification reactions were performed in triplicate in 30 mL of final volume via TaqMan chemistry and with appropriate primers and probes designed with the Primer Express software (Applied Biosystems, CA) using validation and equation as above described. Primers and probes sequences are given in Table 1.

Data reported in Table 1 show that the mtDNA transcript level (COX, 16S rRNA) is significantly decreased by aging in the soleus muscle. ALCAR supplementation upregulates the transcription of the corresponding genes, bringing back the transcript levels to those of the young animals. Consistent results were previously obtained in aged rat cerebral hemispheres and heart. ² The same behavior of mtDNA transcripts is shown by the nuclear gene for a cytochrome c oxidase (COX) subunit (COX IV).

To verify if the mtDNA and nuclear DNA transcript upregulation was due to an ALCAR-mediated activation of the upstream mitochondriogenic cascade, we analyzed, by means of qRT-PCR, the mRNAs level of some factors of the PGC-1α-dependent mitochondrial biogenesis signaling pathway, namely the master factor PGC-1α itself, the TFAM and one of the two mitochondrial transcription factors B (TFB2). ²⁰ In the soleus muscle during aging PGC-1α, the transcription of the TFAM and TFB2 genes was significantly downregulated with respect to the young samples. ALCAR administration for 2 months to aged animals increased the examined transcripts levels with an almost complete reversal of the age-related decrease, suggesting a stimulation of mitochondrial biogenesis.

To estimate whether ALCAR treatment affected also the content of the PGC-1α protein, total proteins from soleus muscles of young (6 months), old (28 months), and old rats (28 months, 2 months of ALCAR treatment) were analyzed by western blotting using goat anti-PGC-1α polyclonal antibody (SC-5816, Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-α actin polyclonal antibody (Sigma-Aldrich Corp., St. Louis, MO) to normalize the PGC-1α data.

The PGC-1α protein level decreased during aging (about 20%). However, the 2 months of ALCAR treatment in 28-month-old rats raised the PGC-1α protein content to a statistically significant value 20% above the young counterpart. This increase might be due to the effect of ALCAR administration on the half-life of the protein itself or, more probably, according to the results of PGC-1α mRNA, to an increased expression of the corresponding gene.