Inhibition of the oxygen sensor PHD2 in the liver improves survival in lactic acidosis by activating the Cori cycle Tomohiro Suharaa,b,1, Takako Hishikia,c,d,e,1, Masataka Kasaharaa,f,1, Noriyo Hayakawaa,c,d, Tomoko Oyaizua,b, Tsuyoshi Nakanishia,g, Akiko Kuboa,d, Hiroshi Morisakib, William G. Kaelin Jr.h,i,2, Makoto Suematsua,d,2, and Yoji Andrew Minamishimaa,d,2 aDepartment of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan; bDepartment of Anesthesiology, Keio University School of Medicine, Tokyo 160-8582, Japan; cTranslational Research Center, Keio University School of Medicine, Tokyo 160-8582, Japan; dJapan Science and Technology Agency, Exploratory Research for Advanced Technology, Suematsu Gas Biology Project, Core Research for Evolutional Science and Technology, Tokyo 160-8582, Japan; eJapan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo 160-8582, Japan; fDepartment of Pharmacology, Tokyo Dental College, Tokyo 101-0061, Japan; gMS Business Unit, Shimadzu Corporation, Kyoto 604-8511, Japan; hDepartment of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02215; and iHoward Hughes Medical Institute, Chevy Chase, MD 20815 Contributed by William G. Kaelin, Jr., August 12, 2015 (sent for review June 22, 2015; reviewed by Ralph J. DeBerardinis) Loss of prolyl hydroxylase 2 (PHD2) activates the hypoxia-inducible Results and Discussion factor-dependent hypoxic response, including anaerobic glycolysis, Inactivation of Phd2 in the Liver Reduces the Blood Lactate Level. which causes large amounts of lactate to be released from cells into PHD2 is the dominant HIF-prolyl hydroxylase in vivo among all the circulation. We found that Phd2-null mouse embryonic fibroblasts three PHDs (4), and inactivating PHD2 alone is sufficient to ac- (MEFs) produced more lactate than wild-type MEFs, as expected, tivate HIF (5), which would enhance lactate efflux from the cells. − − whereas systemic inactivation of PHD2 in mice did not cause hyper- As expected, lactate efflux from Phd2 / mouse embryonic fibro- + + lacticacidemia. This unexpected observation led us to hypothesize blasts (MEFs) was significantly higher than from Phd2 / MEFs that the hypoxic response activated in the liver enhances the Cori (Fig. 1A). We therefore expected that systemic inactivation of – cycle, a lactate glucose carbon recycling system between muscle PHD2 would cause increased lactic acid production and hyper- and liver, and thereby decreases circulating lactate. Consistent with lacticacidemia. To test this, we studied mice in which Phd2 can be this hypothesis, blood lactate levels measured after a treadmill or conditionally inactivated systemically (Phd2-SKO) (5). We first Phd2 lactate tolerance test were significantly lower in -liver-specific confirmed that the expression levels of some mRNAs linked to Phd2-LKO 13 knockout ( ) mice than in control mice. An in vivo C-labeled glycolysis were significantly increased in skeletal muscles of Phd2- lactate incorporation assay revealed that the livers of Phd2-LKO mice 13 SKO mice compared to control mice (Fig. S1A and Table S1). produce significantly more glucose derived from C-labeled lactate Unexpectedly, however, blood lactate levels in Phd2-SKO were than control mice, suggesting that blockade of PHD2 in the liver ame- not higher, and in fact tended to be lower, than in the control liorates lactic acidosis by activating gluconeogenesis from lactate. Phd2 LKO mice, although this difference did not reach statistical significance - mice were resistant to lactic acidosis induced by injection (Fig. 1B). To examine the mechanisms responsible for this un- of a lethal dose of lactate, displaying a significant elongation of sur- expected observation, we measured lactate levels in mice sub- vival. Moreover, oral administration of a PHD inhibitor improved sur- jected to treadmill exercise. Blood lactate levels were significantly vival in an endotoxin shock mice model. These data suggest that PHD2 is a potentially novel drug target for the treatment of lactic acidosis, which is a serious and often fatal complication observed in Significance some critically ill patients. When oxygen availability becomes limited, organs and cells ac- hypoxic response | PHD inhibitor | hyperlactatemia | gluconeogenesis | tivate the hypoxic response to generate energy. This response sepsis releases a large amount of lactate into the circulation as a result of anaerobic glycolysis. However, we found that activating the hypoxic response in the liver by inhibiting the oxygen sensor n metazoans, the cellular enzyme lactate dehydrogenase (LDH) prolyl hydroxylase domain-containing protein 2 (PHD2) en- converts pyruvate to lactate, which is then exported from cells. I hances the uptake of lactate for gluconeogenesis, also known as Lactate efflux is increased under hypoxic conditions, where avail- the Cori cycle, and ameliorates lactic acidosis. Our findings sug- able molecular oxygen is limited. The cellular response to hypoxia is gest that PHD2 serves as a viable drug target for the treat- mainly regulated by the heterodimeric transcription factor hypoxia- ment of life-threatening lactic acidosis, which is frequent inducible factor (HIF), which consists of an unstable alpha subunit complication of severe infectious and ischemic diseases, as well α β (HIF ) and a stable beta subunit (HIF ) (1, 2). HIF prolyl hy- as of biguanide treatment in patients with diabetes with droxylases called PHDs (PHD1–PHD3) (3) target HIFα for ubiq- renal failure. uitin-proteasome-dependent protein degradation under normoxic conditions, lowering HIF levels and suppressing HIF signaling. Author contributions: T.S., T.H., M.K., W.G.K., M.S., and Y.A.M. designed research; T.S., Under hypoxic conditions, PHDs become inactive because they re- T.H., M.K., N.H., T.O., W.G.K., M.S., and Y.A.M. performed research; T.S., T.H., M.K., W.G.K., M.S., and Y.A.M. analyzed data; and T.S., T.H., M.K., T.N., A.K., H.M., W.G.K., M.S., and Y.A.M. quire molecular oxygen for enzymatic activity. This inactivation leads wrote the paper. α to HIF stabilization and activation of HIF signaling. Activated HIF Reviewers included: R.J.D., University of Texas-Southwestern Medical Center. up-regulates genes involved in various events, including erythro- The authors declare no conflict of interest. cytosis, neovascularization, and anaerobic glycolysis. HIF activation 1T.S., T.H., and M.K. contributed equally to this work. enhances anaerobic glycolysis by up-regulating glucose trans- 2To whom correspondence may be addressed. Email: [email protected], porters and glycolytic enzymes, including lactate dehydro- [email protected], or [email protected]. genase A (LDHA), and monocarboxylate transporters, leading This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to enhanced lactate secretion. 1073/pnas.1515872112/-/DCSupplemental. 11642–11647 | PNAS | September 15, 2015 | vol. 112 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1515872112 Downloaded by guest on October 1, 2021 ABC D E p=0.000548 p=2.19x10-19 0.9 p=0.00101 9 9 5 5 p=0.891 0.15 0.8 8 8 4 4 0.7 7 7 p=0.325 0.6 6 6 0.10 3 3 0.5 5 p=0.248 5 0.4 4 4 2 2 0.3 3 3 0.05 0.2 2 2 1 1 Phd2/Actb 0.1 1 1 0 Blood lactate level (mM) 0 Blood lactate level (mM) 0 0 0 0 +/+ -/- Urinary lactate level (mM) Urinary lactate level (mM) Lactate efflux (pmol/h/cell) Phd2 Phd2 Control (n=3) Control (n=3) 0 min 20 min Control (n=7) MEFs MEFs Phd2-SKO (n=3) Phd2-SKO (n=3) Control (n=3) Phd2-SKO (n=3) Phd2-LKO (n=8) FG H p=0.00202 p=0.0396 7 7 80 6 6 10 9 200 p=0.0000253 5 5 70 (mM) 8 4 4 7 150 p=0.405 3 3 60 6 5 * † 2 2 100 50 4 ‡ 1 1 3 Control (n=8) § 50 0 0 40 2 Blood lactate level (mM) Blood lactate level (mM) Phd2-LKO (n=7) Time to exhaustion (min) 1 Control Phd2-LKO 0 Control (n=11) Control (n=11) Blood lactate level 0 (n=11) (n=12) 0 5 10 15 20 Control Phd2-LKO Phd2-LKO (n=12) Phd2-LKO (n=12) Time (min) Blood lactate AUC (mM x min) (n=8) (n=7) + + − − Fig. 1. Inactivation of Phd2 in the liver reduces blood lactate levels. (A) Lactate efflux analysis in Phd2 / and Phd2 / MEFs. Values were from triplicated dishes. Error bars indicate 1 SEM. (B) The blood lactate level in Control and Phd2-SKO mice. Error bars, SEM. (C) The blood lactate level 50 min after the treadmill in Control and Phd2-SKO mice. Error bars, SEM. (D) Urinary lactate measurement. Lactate levels in urine were measured before (Left) and 20 min after (Right) the lactate injection in Control and Phd2-SKO mice. Error bars, SEM. (E) Real-time RT-PCR analysis of Phd2 mRNA in the livers of Control and Phd2-LKO mice. Error bars, SEM. (F) Blood lactate levels in Control and Phd2-LKO mice. Error bars, SEM. (G) Treadmill experiment in Control and Phd2-LKO mice. The blood lactate levels 50 min after the exercise (Left) and time to exhaustion (Right) are shown. Error bars, SEM. (H) Lactate tolerance test. 0.5 mg per gram body weight of lactate was intraperitoneally injected, and the blood lactate levels were measured at the indicated time (Left). Area under the curve (AUC) was also analyzed (Right). Error bars, SEM. *P = 0.0448; †P = 0.000342; ‡P = 0.000118; §P = 2.53 × 10−7 versus Control. lower in Phd2-SKO mice after treadmill exercise compared to in the Phd2-LKO mice after 50 min of treadmill exercise were control mice (Fig.