The Journal of Neuroscience, October 21, 2015 • 35(42):14353–14369 • 14353 Neurobiology of Disease Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase- Lactic Acid Axis Mithilesh Kumar Jha,1 Gyun Jee Song,1 Maan Gee Lee,1 Nam Ho Jeoung,2 Younghoon Go,3 Robert A. Harris,4 Dong Ho Park,5 Hyun Kook,6 In-Kyu Lee,3 and Kyoungho Suk1 1Department of Pharmacology, Brain Science and Engineering Institute, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea, 2Department of Pharmaceutical Science and Technology, Catholic University of Daegu, Gyeongsan, Republic of Korea, 3Department of Internal Medicine, Division of Endocrinology and Metabolism, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea, 4Roudebush Veterans Administration Medical Center and the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, 5Department of Ophthalmology, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea, and 6Department of Pharmacology, Chonnam National University Medical School, Gwangju, 501-746, Republic of Korea Pyruvate dehydrogenase kinases (PDK1–4) are mitochondrial metabolic regulators that serve as decision makers via modulation of pyruvate dehydrogenase (PDH) activity to convert pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Metabolic dysregulation and inflammatory processes are two sides of the same coin in several pathophysiological conditions. The lactic acid surge associated with the metabolic shift has been implicated in diverse painful states. In this study, we investigated the role of PDK- PDH-lactic acid axis in the pathogenesis of chronic inflammatory pain. Deficiency of Pdk2 and/or Pdk4 in mice attenuated complete Freund’s adjuvant (CFA)-induced pain hypersensitivities. Likewise, Pdk2/4 deficiency attenuated the localized lactic acid surge along with hallmarks of peripheral and central inflammation following intraplantar administration of CFA. In vitro studies supported the role of PDK2/4 as promoters of classical proinflammatory activation of macrophages. Moreover, the pharmacological inhibition of PDKs or lactic acid production diminished CFA-induced inflammation and pain hypersensitivities. Thus, a PDK-PDH-lactic acid axis seems to mediate inflammation-driven chronic pain, establishing a connection between metabolism and inflammatory pain. Key words: chronic inflammatory pain; inflammation; macrophages; metabolism; pain; PDK-PDH-lactic acid axis Significance Statement The mitochondrial pyruvate dehydrogenase (PDH) kinases (PDKs) and their substrate PDH orchestrate the conversion of pyru- vate either aerobically to acetyl-CoA or anaerobically to lactate. Lactate, the predominant end product of glycolysis, has recently been identified as a signaling molecule for neuron-glia interactions and neuronal plasticity. Pathological metabolic shift and subsequent lactic acid production are thought to play an important role in diverse painful states; however, their contribution to inflammation-driven pain is still to be comprehended. Here, we report that the PDK-PDH-lactic acid axis constitutes a key component of inflammatory pain pathogenesis. Our findings establish an unanticipated link between metabolism and inflamma- tory pain. This study unlocks a previously ill-explored research avenue for the metabolic control of inflammatory pain pathogenesis. Introduction 2011). Emerging evidence has outlined the crosstalk between in- Chronic inflammation has been identified as a central feature of flammatory processes and metabolic dysregulation under patho- the metabolic syndrome (Hotamisligil, 2006; Lumeng and Saltiel, logical conditions (Palomer et al., 2013). Cells participating in Received May 18, 2015; revised Sept. 14, 2015; accepted Sept. 14, 2015. This work was supported by Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic Author contributions: M.K.J. and K.S. designed research; M.K.J. performed research; G.J.S., M.G.L., N.H.J., Y.G., of Korea Grants A111345 and HI14C3331 and the Basic Science Research Program through the National Research R.A.H., D.H.P., H.K., and I.-K.L. contributed unpublished reagents/analytic tools; M.K.J., G.J.S., N.H.J., Y.G., R.A.H., Foundation funded by the Korean Ministry of Education, Science, and Technology 2008-0062282. D.H.P., H.K., I.-K.L., and K.S. analyzed data; M.K.J. and K.S. wrote the paper. The authors declare no competing financial interests. 14354 • J. Neurosci., October 21, 2015 • 35(42):14353–14369 Jha et al. • PDK-PDH-Lactic Acid Axis in Inflammatory Pain inflammation experience diverse metabolic changes, including a a different reactivity for these three sites, and phosphorylation at shift toward enhanced glucose uptake and glycolysis. Altered me- any site leads to the inhibition of PDH activity. Dysregulation of tabolism may participate in signal-directed programs that pro- the PDH/PDK system has been implicated in life-threatening mote or inhibit inflammation (O’Neill and Hardie, 2013). neurometabolic disorders, including Alzheimer’s disease, trau- Mitochondrial metabolic shift has been identified as a hallmark matic brain injury, brain aging, and glioblastoma (Jha et al., 2012; of highly prevalent diseases, such as obesity/diabetes, cancer Palamiuc et al., 2015). PDK-induced phosphorylation and sub- (Ramos-Nino, 2013), and cardiovascular disease (Nisoli et al., sequent inactivation of PDC result in metabolic shift toward gly- 2007). Mitochondrial malfunction, aberrant glucose metabo- colysis and lactate production, which led us to hypothesize that lism, and the subsequent dysregulation of bioenergetics are con- lactic acid produced under this condition may have tremendous sistent antecedents to the pathophysiology of the nervous system effects on inflammation and pain behaviors. Thus, a PDK-PDH- as well (Jha et al., 2012). Accumulating evidence has implicated lactic acid axis may be a key component that governs chronic the metabolic shift (Joseph and Levine, 2006), reactive oxygen inflammatory pain development. This possibility was tested us- species (Kim et al., 2004), genetic defects (Joseph and Levine, ing Pdk2- and Pdk4-deficient mice and chemical inhibitors of 2010), diabetes (Fernyhough et al., 2010), chemotherapeutics PDK or lactic acid production in the complete Freund’s adjuvant (Bennett, 2010), and HIV infection (Ferrari and Levine, 2010)in (CFA)-injected rodent model of inflammatory pain. Macro- the pathogenesis of painful peripheral neuropathies. These stud- phages in culture were also used to examine the mechanistic re- ies suggest that modulation of mitochondrial function is a prom- lationship between PDKs and proinflammatory phenotypes. ising therapeutic strategy for the management of diverse pain states (Baloh, 2008). Materials and Methods Pain hypersensitivity that results from infection, inflamma- Mouse breeding and maintenance. All experiments were conducted in tion, or peripheral injury to tissue is collectively called inflamma- accordance with approved animal protocols and guidelines established tory pain (Kopach et al., 2012). The development of pain by the Animal Care Committee of Kyungpook National University (ap- hypersensitivity in response to inflammatory insults/stimuli is proval no. KNU-2012–73/66). All efforts were made to minimize the due to peripheral (Basbaum et al., 2009) and central sensitiza- number of animals used and animal suffering. Male wild-type (WT, ϩ ϩ ϩ ϩ Ϫ Ϫ tions (Kuner, 2010). Recruitment of immune/inflammatory cells Pdk2 / Pdk4 / ), Pdk2 knock-out (Pdk2 / ), Pdk4 knock-out Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ in response to inflammatory insult and the subsequent release of (Pdk4 ), and Pdk2/4 double knock-out (DKO, Pdk2 Pdk4 ) proinflammatory/algesic mediators activate peripheral nocicep- mice 8–10 weeks of age were used. Pdk2 KO and Pdk4 KO mice were tors resulting in peripheral sensitization, whereas the long-term generated as previously described (Jeoung et al., 2012). Pdk2 KO mice were crossed with Pdk4 KO mice to produce Pdk2/4 DKO mice. Age- potentiation in the spinal cord is regarded as a unique form of matched WT mice were produced from the C57BL/6J black mice (The central sensitization (Ji et al., 2013). Lactic acid, the predominant Jackson Laboratory) that were used to stabilize the genetic backgrounds end product of glycolysis (Rogatzki et al., 2015), has recently been of the Pdk2 KO and Pdk4 KO mice. Heterozygous animals were crossed identified as a signaling molecule for neuron–glia interactions to generate littermate control animals, which were used to confirm the (Tang et al., 2014) and neuronal plasticity (Yang et al., 2014). findings of behavioral studies. Genotypes were confirmed by PCR of the Lactic acid also contributes to pathological pain by facilitating genomic DNA as described previously (Herbst et al., 2014). Animals were nociceptor activation via lowered pH (Kim et al., 2007). Muscle housed under a 12 h light/dark cycle (lights on 07:00–19:00) at a constant Ϯ ischemia or microvascular dysfunction generates lactic acid and ambient temperature of 23 2°C with food and water provided ad inflammatory mediators that play a crucial role in the develop- libitum. Each individual animal was used in only one experiment. ment of cutaneous tactile allodynia by activating acid sensing ion CFA-induced chronic/persistent inflammation.