Molecular and cellular pharmacologyAspirin may influence cellular energy status
Introduction
Acetylsalicylic acid (ASA) has an analgesic property and serves as an important modulator of cyclooxygenase (COX), and thus the production of thromboxanes (Vane, 1971, Vane and Botting, 2003, Rowlinson et al., 2003). The beneficial actions of ASA have been mainly attributed to this property; ASA is believed to be readily hydrolyzed by enzymes, including paraoxonase 1 (PON1) (Santanam and Parthasarathy, 2006) to salicylic acid (SA). ASA has a short plasma half-life of less than 15 min and the product, SA, has profound biological actions of its own. The half-life of SA exceeds several hours and is concentration-dependent. In addition to its well-known analgesic actions (Williams and Hennekens, 2004, Hennekens et al., 1989, Claria and Serhan, 1995), SA also has been extensively studied as a hydroxyl radical trap (John et al., 1993). It is excreted as a glucuronide and other conjugates (Gutierrez et al., 2006), suggesting that it undergoes metabolic conversion in the liver. However, SA is known to be metabolized by liver microsomes enzymatically as well as non-enzymatically (Grootveld and Halliwell, 1986) to 2,3- and 2,5- dihydroxy benzoic acid (DHBA) by hydroxyl radicals. These dihydroxybenzene derivatives, such as 2,3- or 2,5-DHBA, are expected to autooxidize to form quinones and reactive oxygen species, particularly hydrogen peroxide (H2O2) (Kamble et al., 2013). Reactive oxygen species and H2O2 have been known to be attributed to an increase in the induction of sirtuins (Sirt) such as Sirt1, Sirt3 and Sirt4 (Alcendor et al., 2004, Kwon and Ott, 2008). Recent studies showed that there are few protein substrates for Sirt 2–7 which play an important role in multiple disease-relevant pathways largely encompassing mitochondrial energetics and inflammatory cardiomyopathy. Increased Sirt1 activity would decrease glucose levels, increase the number of mitochondria, regulate insulin sensitivity and lower body weight (Elliott and Jirousek, 2008). Among other sirtuins, Sirt4 is a mitochondrial protein that does not display nicotinamide adenine dinucleotide (NAD)-dependent deacetylase activity, but it utilizes NAD for carrying out adenosine diphosphate (ADP)–ribosylation of glutamate dehydrogenase (GDH) in the mitochondria. Nevertheless, ADP–ribosylation of GDH represses GDH activity by Sirt4, which limits metabolism of glutamate and glutamine to generate adenosine triphosphate (ATP) (Haigis et al., 2006). Thus, we tested the ability of ASA and SA to generate H2O2 to induce Sirt4, Nrf2, Tfam, UCP1, eNOS and STAT3 genes responsible for mitochondrial function and liver×receptorα, farnesoid×receptor and PPARα genes involved in lipogenesis and cholesterol homeostasis.
Section snippets
Chemicals
ASA and all other chemicals were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO). HepG2 cells were purchased from the American Type Culture Collection (ATCC), (Manassas, VA). All other reagents used were of analytical grade. PCR primers and cell culture reagents were obtained from Invitrogen (Carlsbad, CA).
Cell culture and treatment by ASA
HepG2 cells were cultured in advanced DMEM (Invitrogen, Carlsbad, CA), supplemented with 10% FBS, 1% l-glutamine, and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA) and were
Sirt4, Nrf2, Tfam, UCP1 and STAT3 gene expression in HepG2 cells
Sirt4 is a mitochondrial protein that utilizes NAD+ for carrying out ADP–ribosylation of glutamate dehydrogenase (GDH) in mitochondria (Hai et al., 2009). Treatment of HepG2 cells with 50 µM ASA for 48 h resulted in an increase in Sirt4 (4.55±1.8 fold) as compared to control (Fig. 1A). Nrf2 is a nuclear respiratory factor known for transcriptional control of mitochondrial genes. It is essential in mitochondrial biogenesis (Elliott and Jirousek, 2008, Repa and Mangelsdorf, 2000). Increased Nrf2
Discussion
In our previous studies, we have demonstrated the induction of Sirt1 and PGC1α by ASA with a suggested mechanism showing the generation of O2−. and H2O2 as the inducer (Kamble et al., 2013). We have also reported that ASA hydrolysis to SA by PON1 and further hydroxylation to 2,3-DHBA by Ah receptor occurs via Cyp1A1 inducing Sirt1 and other antioxidants. A conclusion from previous studies is that induction of these transcription factors would coordinate in regulation of various metabolic
Acknowledgments
This work is supported by NIH RO-1 (Grant no. 60015108) on “Novel mechanisms by which aspirin might protect against atherosclerosis.”
The authors thank Dr. Kathryn Young Burge for her assistance in preparation of manuscript.
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