Mitochondrial Lactate Metabolism Is Involved in Antioxidative Defense in Human Astrocytoma Cells Joseph Lemire, Christopher Auger, Ryan Mailloux, and Vasu D. Appanna* Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada Although lactate has traditionally been known to be an end product of anaerobic metabolism, recent studies have revealed its disparate biological functions. Oxidative energy production and cell signaling are two important roles assigned to this monocarboxylic acid. Here we demonstrate that mitochondrial lactate metabolism to pyruvate mediated by lactate dehydrogenase (LDH) in a human astrocytic cell line is involved in antioxidative defense. The pooling of this a-ketoacid helps to detoxify reactive oxygen species, with the concomitant formation of acetate. In-gel activity assays following blue native PAGE electrophoresis were utilized to demonstrate the increase in mitochondrial LDH activity coupled to the decrease in pyruvate dehydrogenase activity in the cells challenged by oxidative stress. The enhanced production of pyruvate with the concomitant formation of acetate in astrocytoma cells was monitored by high-performance liquid chromatography. The ability of pyruvate to fend off oxidative stress was visualized by fluorescence micros- copy with the aid of the dye 2 0 ,7 0 -dichlorodihydrofluores- cein diacetate. Immunoblotting helped confirm the presence of elevated levels of LDH in cells exposed to oxidative stress, and recovery experiments were per- formed with pyruvate to diminish the oxidative burden on the astrocytoma. The acetate, generated as a conse- quence of the antioxidative attribute of pyruvate, was subsequently channeled toward the production of lipids, a process facilitated by the upregulation in activity of acetyl-CoA synthetase and acetyl-CoA carboxylase, as demonstrated by in-gel activity assays. The mitochondrial lactate metabolism mediated by LDH appears to play an important role in antioxidative defence in this astrocytic system. V C 2014 Wiley Periodicals, Inc. Key words: astrocytes; lactate dehydrogenase; metabo- lism; pyruvate; reactive oxygen species Recent discoveries on the function of lactate in biology have expanded the role of this monocarboxylic acid from being an end product of anaerobic metabolism to its participation in oxidative energy production (Brooks, 2007; Lemire et al., 2008; De Bari et al., 2010; Cruz et al., 2012), cell regulation (Brooks, 2009; Ber- gersen and Gjedde, 2012; Gohil and Brooks, 2012; Latham et al., 2012; Rinholm and Bergersen, 2012), and long-term potentiation (LTP) for memory consolidation (Bezzi and Volterra, 2011; Suzuki et al., 2011). Lactate is a member of the monocarboxylate family, which includes acetate and pyruvate. Acetate has received recent atten- tion as an astrocyte-specific energy substrate (Deelchand et al., 2009; Wyss et al., 2011; K€ unnecke, 2012) and likely contributes to glial lipid production, because it is a source of acetyl-CoA. In neurons, acetate can be pro- duced through pyruvate from astrocyte-derived lactate or through glycolysis. Acetyl-CoA, in these cells, can be used as a source of energy, for fatty acid synthesis, or for the synthesis of the cholinergic neurotransmitter acetyl- choline (Szutowicz et al., 2013). Pyruvate, an a-keto acid, is known to be involved in a variety of metabolic pathways. However, under oxidative stress, pyruvate is also known to serve a vital role as an antioxidant in numerous systems (Desagher et al., 1997; Mallet and Sun, 2003; Knott et al., 2006). Aluminum is the most abundant metal in the earth’s crust and has increased in bioavailability as a result of anthropogenic activities such as industrial utilization and soil acidification (Exley, 2009). One of the main modes of toxicity for Al is the induction of a pro-oxidant state. This state is promoted by Al interfering with iron (Fe) homeostasis, which in turn triggers the formation of reactive oxygen species (ROS; Exley, 2004). Al can also interfere with calcium, and magnesium metabolism, as Contract grant sponsor: Laurentian University; Contract grant sponsor: Northern Ontario Heritage Foundation; Contract grant sponsor: NSERC-Postdoctoral Fellowship (to J.L.); Contract grant sponsor: NSERC Postgraduate Scholarship (to C.A.). *Correspondence to: Vasu D. Appanna, Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada P3E 2C6. E-mail: vappanna@laurentian.ca J. Lemire’s current address is Department of Biological Sciences, Univer- sity of Calgary, 2500 University Dr. N.W., Calgary, Alberta, Canada T2N 1N4. R. Mailloux’s current address is Department of Biochemistry, Microbiol- ogy and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. Received 14 July 2013; Revised 28 October 2013; Accepted 4 November 2013 Published online 22 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23338 V C 2014 Wiley Periodicals, Inc. Journal of Neuroscience Research 92:464–475 (2014)